RESUMEN
TGF-ß and Wnt/ß-catenin signaling pathways are known to be essential for the development of periodontal tissue. In this study, we examined the crosstalk between TGF-ß and Wnt/ß-catenin signaling in ligament-fibroblastic differentiation of human periodontal ligament cells (hPDLCs). TGF-ß1 treatment significantly increased the expression of ligament-fibroblastic markers, but such expression was preventing by treatment with SB431542, a TGF-ß type I receptor inhibitor. As well as phosphorylation of Smad3, TGF-ß1 increased ß-catenin activation. The depletion of ß-catenin reduced the expression of ligament-fibroblastic markers, suggesting that ß-catenin is essential for ligament differentiation. The effect of TGF-ß1 on ß-catenin activation did not seem to be much correlated with Wnt stimuli, but endogenous DKK1 was suppressed by TGF-ß1, indicating that ß-catenin activation could be increased much more by TGF-ß1. In addition to DKK1 suppression, Smad3 phosphorylation by TGF-ß1 facilitated the nuclear translocation of cytoplasmic ß-catenin. In contrast to ligament-fibroblastic differentiation, inhibition of TGF-ß1 signaling was needed for cementoblastic differentiation of hPDLCs. BMP7 treatment accompanied by inhibition of TGF-ß1 signaling had a synergistic effect on cementoblastic differentiation. In conclusion, ß-catenin activation by TGF-ß1 caused ligament-fibroblastic differentiation of hPDLCs, and the presence of TGF-ß1 stimuli basically determined whether hPDLCs are differentiated into ligament progenitor or cementoblasts.
RESUMEN
2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vß13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.
Asunto(s)
Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Transgenes/genética , Animales , Southern Blotting , Cromosomas , Expresión Génica , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Basic fibroblast growth factor (bFGF or FGF-2) is widely used to modulate the proliferation and differentiation of certain cell types. An expression and purification system for recombinant human FGF-2 in Escherichia coli was established for the purpose of securing a continuous supply of this protein. The purified recombinant FGF-2 significantly increased the population of human embryonic stem cells. The optimal concentrations of FGF-2 for cell proliferative induction in various adult stem cells including human dental pulp stem cells, full term human periodontal ligament stem cells, human gingival fibroblasts, mesenchymal stem cells, and osteogenic oseosarcoma were established in a dose-dependent manner. When cells were treated with recombinant FGF-2 for 6 days before osteogenic induction, the mRNA expression of the bone markers was upregulated in cells originated from human dental pulp tissue, indicating that pretreatment with FGF-2 during culture increase stem cell/progenitor population and osteogenic potential.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Pulpa Dental/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Proteínas Recombinantes/farmacología , Células Madre/citologíaRESUMEN
B-Cell receptor-associated protein 31 (BAP31) regulates the export of secreted membrane proteins from the endoplasmic reticulum (ER) to the downstream secretory pathway. Previously, we generated a monoclonal antibody 297-D4 against the surface molecule on undifferentiated human embryonic stem cells (hESCs). Here, we found that 297-D4 antigen was localized to pluripotent hESCs and downregulated during early differentiation of hESCs and identified that the antigen target of 297-D4 was BAP31 on the hESC-surface. To investigate the functional role of BAP31 in hESCs, BAP31 expression was knocked down by small interfering RNA. BAP31 depletion impaired hESC self-renewal and pluripotency and drove hESC differentiation into multicell lineages. BAP31 depletion hindered hESC proliferation by arresting cell cycle at G0/G1 phase and inducing caspase-independent cell death. Interestingly, BAP31 depletion reduced hESC adhesion to extracellular matrix (ECM). Analysis of cell surface molecules showed decreased expression of epithelial cell adhesion molecule (EpCAM) in BAP31-depleted hESCs, while ectopic expression of BAP31 elevated the expression of EpCAM. EpCAM depletion also reduced hESC adhesion to ECM, arrested cell cycle at G0/G1 phase and induced cell death, producing similar effects to those of BAP31 depletion. BAP31 and EpCAM were physically associated and colocalized at the ER and cell surface. Both BAP31 and EpCAM depletion decreased cyclin D1 and E expression and suppressed PI3K/Akt signaling, suggesting that BAP31 regulates hESC stemness and survival via control of EpCAM expression. These findings provide, for the first time, mechanistic insights into how BAP31 regulates hESC stemness and survival via control of EpCAM expression.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/metabolismo , Caspasas/metabolismo , Adhesión Celular , Muerte Celular , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Molécula de Adhesión Celular Epitelial , Matriz Extracelular/metabolismo , Fase G1 , Humanos , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Fase de Descanso del Ciclo Celular , Transducción de SeñalRESUMEN
Many studies have shown that persistent infections of bacteria promote carcinogenesis and metastasis. Infectious agents and their products can modulate cancer progression through the induction of host inflammatory and immune responses. The presence of circulating tumor cells (CTCs) is considered as an important indicator in the metastatic cascade. We unintentionally produced a monoclonal antibody (MAb) CA27 against the mycoplasmal p37 protein in mycoplasma-infected cancer cells during the searching process of novel surface markers of CTCs. Mycoplasma-infected cells were enriched by CA27-conjugated magnetic beads in the peripheral blood mononuclear cells in patients with hepatocellular carcinoma (HCC) and analyzed by confocal microscopy with anti-CD45 and CA27 antibodies. CD45-negative and CA27-positive cells were readily detected in three out of seven patients (range 12-30/8.5 ml blood), indicating that they are mycoplasma-infected circulating epithelial cells. CA27-positive cells had larger size than CD45-positive hematological lineage cells, high nuclear to cytoplasmic ratios and irregular nuclear morphology, which identified them as CTCs. The results show for the first time the existence of mycoplasma-infected CTCs in patients with HCC and suggest a possible correlation between mycoplasma infection and the development of cancer metastasis.
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Antígenos Bacterianos/sangre , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/complicaciones , Infecciones por Mycoplasma/patología , Células Neoplásicas Circulantes/inmunología , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Línea Celular , Femenino , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Infecciones por Mycoplasma/sangre , Infecciones por Mycoplasma/inmunología , Células Neoplásicas Circulantes/patologíaRESUMEN
Self-renewal and pluripotency of human embryonic stem cells (hESCs) are a complex biological process for maintaining hESC stemness. However, the molecular mechanisms underlying these special properties of hESCs are not fully understood. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a multifunctional RNA-binding protein whose expression is related to cell proliferation and carcinogenesis. In this study, we found that hnRNP A2/B1 expression was localized to undifferentiated hESCs and decreased upon differentiation of hESCs. hnRNP A2/B1 knockdown reduced the number of alkaline phosphatase-positive colonies in hESCs and led to a decrease in the expression of pluripotency-associated transcription factors OCT4, NANOG, and SOX2, indicating that hnRNP A2/B1 is essential for hESC self-renewal and pluripotency. hnRNP A2/B1 knockdown increased the expression of gene markers associated with the early development of three germ layers, and promoted the process of epithelial-mesenchymal transition, suggesting that hnRNP A2/B1 is required for maintaining the undifferentiated and epithelial phenotypes of hESCs. hnRNP A2/B1 knockdown inhibited hESC proliferation and induced cell cycle arrest in the G0/G1 phase before differentiation via degradation of cyclin D1, cyclin E, and Cdc25A. hnRNP A2/B1 knockdown increased p27 expression and induced phosphorylation of p53 and Chk1, suggesting that hnRNP A2/B1 also regulates the G1/S transition of hESC cell cycle through the control of p27 expression and p53 and Chk1 activity. Analysis of signaling molecules further revealed that hnRNP A2/B1 regulated hESC proliferation in a PI3K/Akt-dependent manner. These findings provide for the first time mechanistic insights into how hnRNP A2/B1 regulates hESC self-renewal and pluripotency.
Asunto(s)
Células Madre Embrionarias/citología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Técnicas de Cocultivo , Regulación hacia Abajo , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fase G1/fisiología , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Ratones , Células Madre Pluripotentes/metabolismo , Fase S/fisiología , TransfecciónRESUMEN
Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.
Asunto(s)
Aberraciones Cromosómicas , Proteínas de Unión al ADN/metabolismo , Linfoma/genética , Linfoma/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Apoptosis , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/genética , Reordenamiento Génico de Linfocito T/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Linfoma/inmunología , Linfoma/metabolismo , Ratones , Ratones Noqueados , Eliminación de Secuencia/genética , Cariotipificación Espectral , Linfocitos T/citología , Timo/citología , Timo/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. RESULTS: A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. CONCLUSIONS: This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.
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Vectores Genéticos/metabolismo , Anticuerpos de Cadena Única/metabolismo , Animales , Unión Competitiva , Epítopos/inmunología , Citometría de Flujo , Vectores Genéticos/genética , Células HEK293 , Células Hep G2 , Humanos , Inmunoprecipitación , Ratones , Biblioteca de Péptidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Tetraspanina 29/genética , Tetraspanina 29/inmunología , Tetraspanina 29/metabolismo , TransfecciónRESUMEN
Despite the recent identification of surface markers of undifferentiated human embryonic stem cells (hESCs), the crucial cell-surface molecules that regulate the self-renewal capacity of hESCs remain largely undefined. Here, we generated monoclonal antibodies (MAbs) that specifically bind to undifferentiated hESCs but not to mouse embryonic stem cells. Among these antibodies, we selected a novel MAb, 4-63, and identified its target antigen as the L1 cell adhesion molecule (L1CAM) isoform 2. Notably, L1CAM expressed in hESCs lacked the neuron-specific YEGHH and RSLE peptides encoded by exons 2 and 27, respectively. L1CAM colocalized with hESC-specific cell-surface markers, and its expression was markedly downregulated on differentiation. Stable L1CAM depletion markedly decreased hESC proliferation, whereas L1CAM overexpression increased proliferation. In addition, the expression of octamer-binding transcription factor 4, Nanog, sex-determining region Y-box 2, and stage-specific embryonic antigen (SSEA)-3 was markedly downregulated, whereas lineage-specific markers and SSEA-1 were upregulated in L1CAM-depleted hESCs. Interestingly, the actions of L1CAM in regulating the proliferation and differentiation of hESCs were exerted predominantly through the fibroblast growth factor receptor 1 signaling pathway. Taken together, our results suggest that L1CAM is a novel cell-surface molecule that plays an important role in the maintenance of self-renewal and pluripotency in hESCs.
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Células Madre Embrionarias/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores/química , Línea Celular , Proliferación Celular , Células Madre Embrionarias/citología , Exones , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunoprecipitación , Ratones , Proteína Homeótica Nanog , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Análisis de Secuencia de Proteína , Transducción de Señal , Antígenos Embrionarios Específico de Estadio/genética , Antígenos Embrionarios Específico de Estadio/metabolismoRESUMEN
Human pluripotent stem cells (hPSCs) exist in at least two distinct states in mammals: naïve pluripotency that represents several molecular characteristics in pre-implantation epiblast and primed pluripotency that corresponds to cells poised for differentiation in post-implantation epiblast. To identify and characterize the surface molecules that are necessary for the maintenance of naïve hPSCs, we generated a panel of murine monoclonal antibodies (MAbs) specific to the naïve state of hPSCs. Flow cytometry showed that N1-A4, one of the MAbs, bound to naïve hPSCs but not to primed hPSCs. Cell surface biotinylation and immunoprecipitation analysis identified that N1-A4 recognized heat shock protein 60 (HSP60) expressed on the surface of naïve hPSCs. Quantitative polymerase chain reaction (qPCR) analysis showed that HSP60 expression was rapidly downregulated during the embryoid body (EB) differentiation of primed hPSCs. HSP60 knockdown led to a decrease in the expression of pluripotency genes in primed hPSCs. HSP60 depletion also led to a decrease in the expression of pluripotency genes and representative naïve-state-specific genes in naïve hPSCs. Taken together, the results suggest that HSP60 is downregulated during differentiation of hPSCs and is required for the maintenance of pluripotency genes in both primed and naïve hPSCs, suggesting that HSP60 is a regulator of hPSC pluripotency and differentiation.
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Chaperonina 60 , Células Madre Pluripotentes , Animales , Diferenciación Celular , Chaperonina 60/genética , Chaperonina 60/metabolismo , Cuerpos Embrioides , Estratos Germinativos , Humanos , Mamíferos , RatonesRESUMEN
Lung cancer is the most frequent cause of cancerassociated mortality worldwide. Upregulation of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) has been reported in nonsmall cell lung cancer (NSCLC) cells, but its contribution to NSCLC remains poorly understood. hnRNPA2/B1 is involved in carcinogenesis by interacting with a number of proteins; however, little is known about its interaction with p53. The results of the present study revealed that hnRNPA2/B1 expression levels were upregulated in NSCLC cells under tumorsphere culture conditions and cisplatin treatment compared with those in cells under the adherent condition and dimethyl sulfoxide treatment, respectively, suggesting that hnRNPA2/B1 expression is induced under stress conditions. hnRNPA2/B1 knockdown decreased the number and size of NSCLC cell colonies in a clonogenic survival assay and led to a decreased migratory potential of NSCLC cells, suggesting that hnRNPA2/B1 may promote the survival, proliferation and migration of NSCLC cells. hnRNPA2/B1 knockdown induced G0/G1 phase arrest in NSCLC cells through cyclin E degradation and phosphorylation of cyclindependent kinase 2. In addition, hnRNPA2/B1 knockdown inhibited extracellular signalregulated kinase (ERK)1/2 phosphorylation, suggesting that hnRNPA2/B1 may promote the G1/S phase transition in NSCLC cells through ERK signaling. hnRNPA2/B1 knockdown resulted in increased expression levels of p21 and p27 in NSCLC cells, as well as p53 induction and phosphorylation. Additionally, hnRNPA2/B1 knockdown inhibited human double minute 2 protein (HDM2) stability and phosphorylation, whereas overexpression of hnRNPA2 induced the opposite effects. These results suggested that hnRNPA2/B1 may promote the survival, proliferation and migration of NSCLC cells through preventing the activation of p53, which is induced by ERKmediated HDM2 activation. The results of the present study also indicated that the components of the hnRNPA2/B1/ERK/p53/HDM2 signaling pathway may be novel potential molecular targets for the treatment of patients with NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Cisplatino/farmacología , Dimetilsulfóxido/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: The differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis. METHODS: To study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-ß1 (TGF-ß1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-ß1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR. RESULTS: The binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin α2, α3, and αV, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin α2 was drastically downregulated, but the expression of integrin α3 and αV was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin α3 and αV was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin αV-high hMSCs have a greater osteogenic potential than integrin αV-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin αV is more dramatically induced even in integrin αV-low hMSCs. CONCLUSION: These findings suggest that integrin α3 and αV induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.
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Células Madre Mesenquimatosas , Osteogénesis , Diferenciación Celular , Células Cultivadas , Humanos , Integrina alfa2 , OsteoblastosRESUMEN
BACKGROUND: Odontoblast is a unique progenitor that plays a role in dentin formation. So far, the dentinogenic differentiation of dental pulp stem cells and the role of surface molecules of odontoblasts in dentinogenesis are not well known yet. In this study, we obtained odontoblast-like cells from human dental pulp cells and screened odontoblast-specific cell surface antigens by decoy immunization. METHODS: Through decoy immunization with intact odontoblast-like cells derived from human dental pulp cells, we constructed 12 monoclonal antibodies (mAbs) of IgG type, and their binding affinities for cell surface of odontoblast-like cells were analyzed by flow cytometry. Immunoprecipitation, mass spectrometry, and immunohistochemistry were performed to demonstrate odontoblast-specific antigens. Odontoblasts were sorted by these mAbs using magnetic-activated cell sorting system, and their mineralization efficiency was increased after sorting. RESULTS: We constructed 12 mAbs of IgG type, which had a strong binding affinity for cell surface antigens of odontoblast-like cells. In human adult tooth, these mAbs accumulated in the odontoblastic layer between dentin and pulp and in the perivascular region adjacent to the blood vessels in the pulp core. Cell surface expression of the antigenic molecules was increased during odontogenic cytodifferentiation and decreased gradually as dentinogenic maturation progressed. Proteomic analysis showed that two representative antigenic molecules, OD40 and OD46, had the potential to be components for cell adhesion and extracellular matrix structures. CONCLUSION: These results suggest that mAbs will be useful for detecting and separating odontoblasts from the primary pulp cells and other lineage cells and will provide information on the structures of extracellular matrix and microenvironment that appears during the dentinogenic differentiation.
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Células Madre Adultas/metabolismo , Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Pulpa Dental/metabolismo , Odontoblastos/metabolismo , Adulto , Células Madre Adultas/citología , Pulpa Dental/citología , Humanos , Odontoblastos/citologíaRESUMEN
Progesterone receptor membrane component1 (PGRMC1) is a heme-binding protein involved in cancers and Alzheimer's disease. PGRMC1 consists of a short N-terminal extracellular or luminal domain, a single membrane-spanning domain, and a long cytoplasmic domain. Previously, we generated two monoclonal antibodies (MAbs) 108-B6 and 4A68 that recognize cell surface-expressed PGRMC1 (csPGRMC1) on human pluripotent stem cells and some cancer cells. In this study, flow cytometric analysis found that an anti-PGRMC1 antibody recognizing the N-terminus of PGRMC1 could not bind to csPGRMC1 on cancer cells, and 108-B6 and 4A68 binding to csPGRMC1 was inhibited by trypsin treatment, suggesting that the epitopes of 108-B6 and 4A68 are trypsin-sensitive. To examine the epitope specificity of 108-B6 and 4A68, glutathione-S-transferase (GST)-fused PGRMC1 mutants were screened to identify the epitopes targeted by the antibodies. The result showed that 108-B6 and 4A68 recognized C-terminal residues 183-195 and 171-182, respectively, of PGRMC1, where trypsin-sensitive sites are located. A polyclonal anti-PGRMC1 antibody raised against the C-terminus of PGRMC1 could also recognized csPGRMC1 in a trypsin-sensitive manner, suggesting that the C-terminus of csPGRMC1 is exposed on the cell surface. This finding reveals that csPGRMC1 has a non-conventional plasma membrane topology, which is different from that of intracellular PGRMC1.
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Anticuerpos Monoclonales/inmunología , Membrana Celular/metabolismo , Mapeo Epitopo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptores de Progesterona/inmunología , Receptores de Progesterona/metabolismo , Línea Celular , Humanos , Espacio Intracelular/metabolismo , Proteínas de la Membrana/química , Receptores de Progesterona/químicaRESUMEN
Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer death worldwide. To study how mycoplasma infection affects HCC progression, we investigated the characteristics of mycoplasma-infected tumor tissues and circulating tumor cells (CTCs) in HCC patients. The mycoplasmal membrane protein p37 showed significant correlations with higher histologic stages and vascular invasion and predicted poor disease-free survival of HCC patients. p37-positive CTCs were detected in 42 out of 47 HCC patients (89%). p37-positive circulating cells were also detected in 4 out of 10 healthy donors (40%), and all were epithelial cell adhesion molecule (EpCAM)-positive. In HCC patients, most of p37-negative CTCs (95%) showed intermediate phenotype with neither EpCAM nor vimentin expression, but p37-positive CTCs were EpCAM-positive (44%), vimentin-positive (32%), and both negative (24%), suggesting that EpCAM-positive CTCs are enriched with mycoplasma infection. Mycoplasma infection promoted migratory capacity of HCC cells with increased expression of EpCAM. Immunoprecipitation analysis revealed that p37 associates with EpCAM. The results suggest that mycoplasma infection promotes tumor progression in HCC patients via interaction of the mycoplasmal p37 and EpCAM.
Asunto(s)
Antígenos Bacterianos/metabolismo , Carcinoma Hepatocelular/microbiología , Molécula de Adhesión Celular Epitelial/metabolismo , Neoplasias Hepáticas/microbiología , Infecciones por Mycoplasma/metabolismo , Mycoplasma hyorhinis/metabolismo , Células A549 , Antígenos Bacterianos/biosíntesis , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Células Hep G2 , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Infecciones por Mycoplasma/sangre , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/patología , Células Neoplásicas CirculantesRESUMEN
In a previous study, we generated a murine hepatitis B virus (HBV)-neutralizing monoclonal antibody (mAb), KR127, that binds to an epitope (amino acids 37-45, NSNNPDWDF) of the preS1 antigen. Furthermore, an epitope tag, S1 (NANNPDWDF), was developed for protein tagging. The aim of the present study was to develop a high-affinity antibody to the same preS1 epitope. Mice were immunized with the N-terminal domain of human thrombopoietin fused to the S1 tag (nTPO-S1), and a phage-displayed chimeric Fab library was constructed and screened by panning against nTPO-S1. A high-affinity antibody (3-34) was selected that binds to the preS1 antigen. The IgG molecules of 3-34 showed approximately nine-fold higher affinity (K(D) 1.2 nM) for preS1 compared with KR127 (K(D) 10.4 nM), competed with KR127 for binding to the epitope, and bound to HBV particles. This study provides a simple and efficient way to develop a high-affinity antibody to a defined epitope by phage display of an immune antibody library.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Epítopos , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Biblioteca de Péptidos , Precursores de Proteínas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Trombopoyetina/inmunologíaRESUMEN
Previously, we constructed a humanized antibody (HuS10) that binds to the common a antigenic determinant on the S protein of HBV. In this study, we evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was intravenously administered with a single dose of HuS10, followed by intravenous challenge with the adr subtype of HBV, while a control chimpanzee was only challenged with the virus. The result showed that the control chimpanzee was infected by the virus, and thus serum HBV surface antigen (HBsAg) became positive from the 14(th) to 20(th) week and actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 19(th) and 23(rd) week, respectively. However, in the case of the study chimpanzee, serum HBsAg became positive from the 34(th) to 37(th) week, while actively acquired serum anti-HBc and anti-HBs antibodies appeared from the 37(th) and 40(th) week, respectively, indicating that HuS10 neutralized the virus in vivo and thus delayed the HBV infection. This novel humanized antibody will be useful in the immunoprophylaxis of HBV infection.
Asunto(s)
Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Pan troglodytes/inmunología , Pan troglodytes/virología , Animales , Células CHO , Cricetinae , Cricetulus , Hepatitis B/sangre , Hepatitis B/inmunología , Hepatitis B/virología , Anticuerpos contra la Hepatitis B/sangre , Pruebas de Neutralización , Pan troglodytes/sangreRESUMEN
BACKGROUND: Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. METHODS: Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. RESULTS: The mAb from hybridoma clone KH7-41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7-33 mAb detected only MMW (hexamer) adiponectin. The KH4-8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4-8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7-33 and KH4-8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. CONCLUSIONS: mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.
Asunto(s)
Adiponectina/sangre , Anticuerpos Monoclonales/administración & dosificación , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Adiponectina/antagonistas & inhibidores , Anciano , Animales , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Persona de Mediana Edad , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/sangreRESUMEN
Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional heme-binding protein involved in various diseases, including cancers and Alzheimer's disease. Previously, we generated two monoclonal antibodies (MAbs) 108-B6 and 4A68 against surface molecules on human pluripotent stem cells (hPSCs). Here we show that PGRMC1 is the target antigen of both MAbs, and is predominantly expressed on hPSCs and some cancer cells. PGRMC1 is rapidly downregulated during early differentiation of hPSCs. Although PGRMC1 knockdown leads to a spread-out morphology and impaired self-renewal in hPSCs, PGRMC1 knockdown hPSCs do not show apoptosis and autophagy. Instead, PGRMC1 knockdown leads to differentiation of hPSCs into multiple lineage cells without affecting the expression of pluripotency markers. PGRMC1 knockdown increases cyclin D1 expression and decreases Plk1 expression in hPSCs. PGRMC1 knockdown also induces p53 expression and stability, suggesting that PGRMC1 maintains hPSC self-renewal through suppression of p53-dependent pathway. Analysis of signaling molecules further reveals that PGRMC1 knockdown promotes inhibitory phosphorylation of GSK-3ß and increased expression of Wnt3a and ß-catenin, which leads to activation of Wnt/ß-catenin signaling. The results suggest that PGRMC1 suppresses the p53 and Wnt/ß-catenin pathways to promote self-renewal and inhibit early differentiation in hPSCs.
Asunto(s)
Autorrenovación de las Células/fisiología , Proteínas de la Membrana/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores de Progesterona/metabolismo , Vía de Señalización Wnt/fisiología , Apoptosis/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Proliferación Celular/fisiología , Regulación hacia Abajo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Células Madre Embrionarias Humanas , Humanos , Fosforilación , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMEN
To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the V beta 13 promoter was either deleted (P13(-/-)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(-/-) mice, cleavage, rearrangement, and transcription of V beta 13, but not the flanking V beta gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of V beta 13 rearrangement was less severe and was not associated with any apparent reduction in V beta 13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal V beta 13-recombination signal sequence junction and V beta 13 coding joint formation of both wild-type and mutant V beta 13 alleles. However, a low level of aberrant V beta 13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated V beta gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant V beta cleavages during allelic exclusion.