RESUMEN
The epigenetic regulation for gene expression determines cell plasticity. Oral squamous cell carcinoma (SCC) exhibits bidirectional cell plasticity, i.e. epithelial differentiation and epithelial to mesenchymal transition (EMT). The epigenetic regulator LSD1 is a histone H3-specific demethylase to which chemical inhibitors for its activity had been developed as an anti-cancer therapeutics. The bidirectional plasticity of the oral SCC cell line OM-1 had been characterized, but it remained unclear how chemical LSD1 inhibitors affect cell plasticity. Here we reported an adverse effect against cancer therapeutics, which was EMT induction in vitro by the chemical LSD1 inhibitor. The LSD1 inhibitor caused EMT-TF ZEB1 in OM-1 to undergo EMT. Furthermore, an additional EMT-TF Snail-dependent partial EMT phenotype in OM-1 progressed to complete EMT in conjunction with LSD1 inhibitor-dependent ZEB1 induction. The promotor activity of ZEB1 was up-regulated under LSD1 inhibition. The regulatory chromatin regions of ZEB1 accumulated histone H3 methylation under the chemical inhibition of LSD1. The LSD1 inhibitor also upregulates epithelial gene expression in vitro; however, the bidirectional effect of LSD1 inhibitor should be considered in cancer therapeutics.
Asunto(s)
Histona Demetilasas , Neoplasias de la Boca , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Línea Celular Tumoral , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/metabolismo , Histonas/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Cancer cells utilize epithelial to mesenchymal transition (EMT) during invasion and metastasis. This program has intermediate cell states with retained epithelial and gained mesenchymal features together, referred to as partial EMT. Histone demethylase LSD1 forms a complex with the EMT master transcription factor Snail to modify histone marks and regulate target gene expression. However, little is known about the formation of this complex during the Snail-dependent transition between partial EMT and EMT. Here we visualized the nuclear complex of Snail and LSD1 as foci signals using proximity ligation assay. We demonstrated that the nuclear foci numbers varied with the transition of exogenous Snail-dependent partial EMT to EMT. Furthermore, we found that long exposure to dexamethasone could revert exogenous Snail-dependent EMT to partial EMT. In this reversion, the nuclear foci numbers also returned to previous levels. Therefore, we concluded that Snail might select partial EMT or EMT by altering its association with LSD1.
RESUMEN
OBJECTIVE: Double-strand (ds) DNA-enveloped viruses can cause oral infection. Our aim is to investigate whether oral mucosal cells participate in immune response against cytosolic dsDNA invasion. METHODS: We examined the response to transfected herpes simplex virus (HSV) dsDNA via intracellular receptors in oral keratinocytes (RT7) and fibroblasts (GT1), and the effect of TNF-α on those responses. RESULTS: Transfected dsDNA increased CXCL10 expression via NF-κB activation in both cell types, while those responses were inhibited by knockdown of RIG-I, an RNA sensor. Although IFI16, a DNA sensor, was expressed in the nuclei of both types, its knockdown decreased transfected dsDNA-induced CXCL10 expression in GT1 but not RT7 cells. IFI16 in GT1 cells was translocated into cytoplasm from nuclei, which was attributed to immune response to cytosolic dsDNA. TNF-α enhanced transfected dsDNA-induced CXCL10, and knockdown of IFI16 decreased TNF-α and dsDNA-driven CXCL10 expression in both RT7 and GT1 cells. Finally, the combination of TNF-α and transfected dsDNA resulted in translocation of IFI16 from nuclei to cytoplasm in RT7 cells. CONCLUSION: RIG-I and IFI16 in oral mucosal cells may play important roles in host immune response against DNA viral infection, while TNF-α contributes to development of an antiviral system via those intracellular receptors.
Asunto(s)
ADN Viral/inmunología , Fibroblastos , Queratinocitos , Simplexvirus/inmunología , Factores de Restricción Antivirales/inmunología , Línea Celular , Quimiocina CXCL10/inmunología , Citoplasma , Fibroblastos/inmunología , Humanos , Inmunidad , Queratinocitos/inmunología , Proteínas Nucleares/inmunología , Fosfoproteínas/inmunología , Receptores de Ácido Retinoico/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
We previously found that microRNAs play major roles in the maintenance of amoeboid-like oral squamous cell carcinoma (OSCC) cells with high expression of CD44 (CD44high ). However, the roles of microRNAs in chemotherapeutic resistance exhibited by CD44high amoeboid-like OSCC cells are unclear. Here, docetaxel-induced apoptosis was examined in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated silicone gel. Amoeboid-like CD44high OSCC cells exhibited robust resistance to docetaxel-induced apoptosis and significant upregulation of miR-224-5p expression compared with epithelial-like CD44high OSCC cells and mesenchymal-like CD44high OSCC cells. The expression of pannexin-1 (PANX1), a channel-forming protein that regulates the release of ATP, was significantly upregulated following transfection of amoeboid-like CD44high OSCC cells with an miR-224-5p inhibitor. These results suggest that miR-224-5p inhibits PANX1 expression. Furthermore, miR-224-5p inhibitor-transfected amoeboid-like CD44high OSCC cells exhibited significant enhancement of the proportion of apoptotic cells; however, this effect was significantly inhibited by knockdown of PANX1 with PANX1 small interfering RNA. Additionally, the miR-224-5p inhibitor-enhanced extracellular ATP levels were significantly reduced by PANX1 knockdown. These findings imply that miR-224-5p plays a vital role in the resistance to docetaxel-induced apoptosis by attenuating PANX1-induced ATP discharge. Moreover, amoeboid-like CD44high OSCC cells may be involved in chemotherapeutic resistance of OSCC.
Asunto(s)
Amoeba , Carcinoma de Células Escamosas , MicroARNs , Neoplasias de la Boca , Apoptosis , Carcinoma de Células Escamosas/genética , Conexinas , Docetaxel/farmacología , Regulación hacia Abajo , Humanos , Receptores de Hialuranos , MicroARNs/genética , Neoplasias de la Boca/genética , Proteínas del Tejido NerviosoRESUMEN
The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.
RESUMEN
TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.
Asunto(s)
Anoctaminas/metabolismo , Canales de Calcio Tipo L/metabolismo , Disferlina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animales , Anoctaminas/análisis , Canales de Calcio Tipo L/análisis , Caveolina 3/análisis , Caveolina 3/metabolismo , Disferlina/análisis , Disferlina/genética , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citologíaRESUMEN
Purpose: The purpose of this study was to investigate the physiological changes of amino acids in the rat retina caused by ocular hypertension.Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Retinas were hydrolyzed with HCl at 108°C to isolate amino acids. Residual amino acids were measured by reverse-phase high-performance liquid chromatography and the total volume of residual amino acids and the ratio of D- and L-amino acids were analyzed. Free D- and L-alanine levels were also measured using two-dimensional HPLC.Results: The amount of retinal alanine decreased in ocular hypertension compared with the control (p < .05, Student's t-test); the amounts of other amino acids did not differ between the two conditions.The D/L ratio of alanine was higher than that of other amino acids. Ocular hypertension reduced the D/L ratio of retinal alanine, while that of other amino acids was unchanged. Ocular hypertension increased the D/L ratio of free alanine.Conclusions: Ocular hypertension reduced the D/L ratio of retinal alanine, presumably due in large part to alanine peptides.
Asunto(s)
Alanina/metabolismo , Hipertensión Ocular/metabolismo , Retina/metabolismo , Animales , Biomarcadores/metabolismo , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Masculino , Hipertensión Ocular/diagnóstico , Ratas , Ratas Wistar , Retina/diagnóstico por imagenRESUMEN
PURPOSE: To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. METHODS: Adult Wistar rats with ocular hypertension were used as an experimental model. D-ß-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-ß-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. RESULTS: D-ß-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and ß, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. CONCLUSION: Ocular hypertension affected the expression of proteins containing D-ß-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.
RESUMEN
Oral keratinocytes provide the first line of host defense against oral candidiasis. We speculated that interactions of fungal cell wall components with oral keratinocytes regulate the stress response against Candida infection and examined the expression of genes induced by heat-killed Candida albicans in oral immortalized keratinocytes using a cDNA microarray technique. Of 24,000 genes revealed by that analysis, we focused on HO-1, a stress-inducible gene, as its expression was increased by both heat-killed and live C. albicans In histological findings, HO-1 expression in the superficial layers of the oral epithelium following Candida infection was elevated compared to that in healthy epithelium. We then investigated fungal cell wall components involved in induction of HO-1 expression and found that ß-glucan-containing particles (ß-GPs) increased its expression. Furthermore, ß-glucan was observed on the surface of both heat-killed C. albicans and Candida cells that had invaded the oral epithelium. Fungal ß-GPs also promoted induction of intracellular reactive oxygen species (ROS), NADPH oxidase activation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation, while those specific inhibitors inhibited the HO-1 expression induced by fungal ß-GPs. Moreover, fungal ß-GPs induced Nrf2 translocation into nuclei via p38 MAPK signaling, while the HO-1 expression induced by fungal ß-GPs was inhibited by Nrf2-specific small interfering RNA (siRNA). Finally, knockdown of cells by HO-1- and Nrf2-specific siRNAs resulted in increased ß-GP-mediated ROS production compared to that in the control cells. Our results show that the HO-1 induced by fungal ß-GPs via ROS/p38 MAPK/Nrf2 from oral keratinocytes may have important roles in host defense against the stress caused by Candida infection in the oral epithelium.
Asunto(s)
Candida albicans/fisiología , Hemo-Oxigenasa 1/genética , Queratinocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , beta-Glucanos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Candidiasis/genética , Candidiasis/metabolismo , Candidiasis/microbiología , Células Cultivadas , Citocinas/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hemo-Oxigenasa 1/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Queratinocitos/microbiología , Mucosa Bucal/inmunología , Mucosa Bucal/metabolismo , Mucosa Bucal/microbiología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) have been shown to be useful markers for identification of cancer stem cells (CSCs). We previously reported that glycogen synthase kinase 3ß (GSK3ß) is involved in regulation of the self-renewal ability of head and neck squamous cell carcinoma (HNSCC) CSCs. The purpose of the present study was to clarify the role of GSK3ß in CD44(high) /ALDH1(high) HNSCC cells. METHODS: Cells with greater expression of CD44 and higher ALDH1 enzymatic activity were FACS sorted from the OM-1 HNSCC cell line. The self-renewal ability of CD44(high) /ALDH1(high) cells was then examined using a tumor sphere formation assay. mRNA expressions of the stem cell markers Sox2, Oct4, and Nanog, as well as GSK3ß were evaluated by real-time RT-PCR. RESULTS: CD44(high) /ALDH1(high) cells exhibited higher tumor sphere forming ability and increased expression of stem cell markers as compared with CD44(high) /ALDH1(low) cells. Interestingly, spindle-shaped cells positive for vimentin were found in the CD44(high) /ALDH1(high) but not the CD44(high) /ALDH1(low) cell population. In addition, the ALDH1 activity and sphere forming ability of CD44(high) /ALDH1(high) cells was significantly inhibited by GSK3ß knockdown. On the other hand, CD44(high) /ALDH1(low) cells exhibited high epidermal growth factor receptor (EGFR) expression and increased cell growth. CONCLUSIONS: Our results show that GSK3ß plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Receptores de Hialuranos/biosíntesis , Isoenzimas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Retinal-Deshidrogenasa/efectos de los fármacos , Familia de Aldehído Deshidrogenasa 1 , Biomarcadores de Tumor/biosíntesis , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Activación Enzimática , Receptores ErbB/biosíntesis , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Receptores de Hialuranos/efectos de los fármacos , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Células Madre Mesenquimatosas/enzimología , Células Madre Mesenquimatosas/metabolismo , Proteína Homeótica Nanog/biosíntesis , Células Madre Neoplásicas/enzimología , Factores de Transcripción de Octámeros/biosíntesis , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/genética , Retinal-Deshidrogenasa/biosíntesis , Retinal-Deshidrogenasa/metabolismo , Factores de Transcripción SOXB2/biosíntesis , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Cancer cells possess unique characteristics such as invasiveness, the ability to undergo epithelial-mesenchymal transition, and an inherent stemness. Cell morphology is altered during these processes and this is highly dependent on actin cytoskeleton remodeling. Regulation of the actin cytoskeleton is, therefore, important for determination of cell fate. Mutations within the TP53 (tumor suppressor p53) gene leading to loss or gain of function (GOF) of the protein are often observed in aggressive cancer cells. Here, we highlight the roles of p53 and its GOF mutants in cancer cell invasion from the perspective of the actin cytoskeleton; in particular its reorganization and regulation by cell adhesion molecules such as integrins and cadherins. We emphasize the multiple functions of p53 in the regulation of actin cytoskeleton remodeling in response to the extracellular microenvironment, and oncogene activation. Such an approach provides a new perspective in the consideration of novel targets for anti-cancer therapy.
Asunto(s)
Citoesqueleto/metabolismo , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Animales , Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Humanos , Integrinas/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: Innate immune response by oral mucosal cells may be the first line of host defense against viral infection. Retinoic acid-inducible gene-I (RIG-I) recognizes viral dsRNA in the cytoplasm, and RIG-I-mediated signaling regulates antiviral type I IFN, and inflammatory chemokine production. Here, we tested the hypothesis that oral mucosal cell participation in host defense against viral infection via RIG-I. METHODS: RIG-I expression was detected in immortalized oral keratinocytes (RT7), oral fibroblasts (GT1) using and RT-PCR and immunohistochemistry. RT7 and GT1 were exposed to dsRNA virus mimic Poly I:C-LMW/LyoVec (PLV). Expression of IFN-ß and CXCL10 via RIG-I was examined by Real-time RT-PCR and ELISA. Phosphorylation of IRF3 and STAT1 were detected by western blotting. RESULTS: RT7 and GT1 constitutively expressed RIG-I in the cytoplasm. Furthermore, PLV increased IFN-ß and CXCL10 productions in both RT7 and GT1 via RIG-I concurrent with phosphorylation of IRF3 and STAT1. PLV-induced CXCL10 production was attenuated by neutralization of IFN-ß and blocking of IFN-α/ß receptor (IFNAR), indicating primal IFN-ß production via the RIG-I-IRF3 axis, which eventually induces CXCL10 production via the IFNAR -STAT1 axis. CONCLUSION: We propose that RIG-I in oral keratinocytes and fibroblasts may cumulatively develop host-defense mechanisms against viral infection in oral mucosa.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Fibroblastos/metabolismo , Queratinocitos/metabolismo , Mucosa Bucal/metabolismo , Línea Celular , Quimiocina CXCL10/metabolismo , Citoplasma/metabolismo , Proteína 58 DEAD Box , Humanos , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Fosforilación/genética , ARN Bicatenario/genética , Receptor de Interferón alfa y beta/metabolismo , Receptores Inmunológicos , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genéticaRESUMEN
TMEM16E/GDD1 has been shown to be responsible for the bone-related late-onset disease gnathodiaphyseal dysplasia (GDD), with the dominant allele (TMEM16E(gdd) ) encoding a missense mutation at Cys356. Additionally, several recessive loss-of-function alleles of TMEM16E also cause late-onset limb girdle muscular dystrophy. In this study, we found that TMEM16E was rapidly degraded via the proteasome pathway, which was rescued by inhibition of the PI3K pathway and by the chemical chaperone, sodium butyrate. Moreover, TMEM16E(gdd) exhibited lower stability than TMEM16E, but showed similar propensity to be rescued. TMEM16E did not exhibit cell surface calcium-dependent chloride channel (CaCC) activity, which was originally identified in TMEM16A and TMEM16B, due to their intracellular vesicle distribution. A putative pore-forming domain of TMEM16E, which shared 39.8% similarity in 98 amino acids with TMEM16A, disrupted CaCC activity of TMEM16A via domain swapping. However, the Thr611Cys mutation in the swapped domain, which mimicked conserved cysteine residues between TMEM16A and TMEM16B, reconstituted CaCC activity. In addition, the GDD-causing cysteine mutation made in TMEM16A drastically altered CaCC activity. Based on these findings, TMEM16E possesses distinct function other than CaCC and another protein-stabilizing machinery toward the TMEM16E and TMEM16E(gdd) proteins should be considered for the on-set regulation of their phenotypes in tissues.
Asunto(s)
Canales de Cloruro/metabolismo , Osteogénesis Imperfecta/metabolismo , Alelos , Secuencia de Aminoácidos , Anoctamina-1 , Anoctaminas , Canales de Cloruro/química , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estabilidad Proteica , Estructura Terciaria de ProteínaRESUMEN
We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and ß5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.
Asunto(s)
Carcinoma de Células Escamosas/patología , Movimiento Celular , Galectina 1/fisiología , Integrina alfa2/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Comunicación Autocrina , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Galectina 1/genética , Galectina 1/farmacología , Humanos , Invasividad Neoplásica , Regulación hacia ArribaRESUMEN
In this study, we found that wounding of a confluent monolayer of squamous cell carcinoma (SCC) cells induced epithelial-mesenchymal transition (EMT) specifically at the edge of the wound. This process required the combined stimulation of TGFß, TNFα, and PDGF-D. Such a combined cytokine treatment of confluent monolayers of the cells upregulated the expression levels of Snail and Slug via PI3K. The PI3K downstream effector, AKT, was dispensable for the upregulation of Snail and Slug, but essential for enabling EMT in response to upregulation of Snail and Slug.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Humanos , Immunoblotting , Inmunohistoquímica , Linfocinas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Factores de Transcripción de la Familia Snail , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll-like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1-10. Interleukin-8 (IL-8) production by RT7 cells was induced by treatment with TLRs 1-9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL-8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor-alpha-induced IL-8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.
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Quimiocina CXCL10/biosíntesis , Fibroblastos/inmunología , Interleucina-8/biosíntesis , Queratinocitos/inmunología , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunología , Bacterias/inmunología , Células Cultivadas , Humanos , Factores Inmunológicos/inmunología , Virus/inmunologíaRESUMEN
We previously identified genes associated with Snail-mediated squamous cell carcinoma (SCC) invasiveness, in which we observed significant elevation of Cyr61 expression. In this study, SCC cell lines overexpressing Cyr61 exhibited constitutive activation of Rho A and upregulated invasiveness without the disruption of homophilic cell attachment. Humoral Cyr61 enhanced further production of endogenous Cyr61 by SCC cells, which stimulated collective cell migration and the development of an invasive tumor nest. We propose a Cyr61-dependent model for the development of invasive SCC nest, whereby a subset of tumor cells that highly produce Cyr61 may direct other tumor cells to undergo collective cell migration, resulting in a formation of primary SCC mass.
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Carcinoma de Células Escamosas/patología , Movimiento Celular , Proteína 61 Rica en Cisteína/metabolismo , Neoplasias de la Boca/patología , Factores de Transcripción/fisiología , Sitios de Unión , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Retroalimentación Fisiológica , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Integrinas/metabolismo , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica , Regiones Promotoras Genéticas , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Receptor for hyaluronan (HA)-mediated motility (RHAMM) was first described as a soluble HA binding protein released by sub-confluent migrating cells. We previously found that RHAMM was highly expressed and plays an important role in proliferation in the human cementifying fibroma (HCF) cell line, which we previously established. HCF is a benign fibro-osseous neoplasm of the jaw and is composed of fibrous tissue containing varying amounts of mineralized material. However, the pathogenesis of HCF is not clear. In this paper, we examined the roles of RHAMM in osteoblastic cells. We generated RHAMM-overexpressing MC3T3-E1 cells and examined the cell proliferation and differentiation of osteoblastic cells. In MC3T3-E1 cells, overexpressing RHAMM was located intracellular and activated ERK1/2. Interestingly, the ERK1/2 activated by RHAMM overexpression promoted cell proliferation and suppressed the differentiation of osteoblastic cells. Our findings strongly suggest that RHAMM may play a key role in the osteoblastic differentiation process. The rupture of balance from differentiation to proliferation induced by RHAMM overexpression may link to the pathogenesis of bone neoplasms such as HCF.
Asunto(s)
Diferenciación Celular , Proteínas de la Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/enzimología , Animales , Anticuerpos/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Ácido Hialurónico/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Osteoblastos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and ß2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ouabaína/farmacología , Ligando RANK/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Vanadatos/farmacología , Fosfatasa Ácida/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Línea Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteínas del Tejido Nervioso/genética , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Ligando RANK/química , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/deficiencia , ATPasa Intercambiadora de Sodio-Potasio/genética , Solubilidad , Fosfatasa Ácida TartratorresistenteRESUMEN
We found a linear correlation between the Prostaglandin E(2) (PGE(2)) amount and the NR4A2 expression in oral squamous cell carcinoma (SCC) tissues through a statistical analysis among 41 clinical cases. In SCC cell lines, PGE(2) receptor (EP) ligation by exogenous PGE(2) promoted the NR4A2 expression in the cAMP/protein kinase A (PKA)-dependent manner. The process required a nature of SCC cell represented by constitutive activated epidermal growth factor receptor (EGFR) family. Targeted inactivation of the EGFRs interfered the PGE(2)-dependent NR4A2 expression. The PGE(2)-dependent NR4A2 induction is essential for the resistance to anti-cancer drug-induced apoptosis especially in SCC cells which showed constitutive EGFRs activity via autocrine epiregulin, a ligand for EGFRs. Conversely, SCC cells which lack epiregulin expression in their nature could gain the ability to promote the NR4A2 expression in response to PGE(2) and attain the resistance to anti-cancer drug-induced apoptosis under the existence of exogenous epiregulin. These findings suggest that susceptibility of SCC to anti-cancer drug could be compromised when PGE(2) was delivered in the microenvironment of SCC cells supported by constitutive EGFR family activities as their nature.