RESUMEN
BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.
Asunto(s)
Movimiento Celular/fisiología , ARN Interferente Pequeño/farmacología , Quinasas Asociadas a rho/fisiología , Línea Celular Tumoral , Neoplasias Esofágicas , Humanos , MicroARNs/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.
Asunto(s)
Humanos , Movimiento Celular/fisiología , ARN Interferente Pequeño/farmacología , Quinasas Asociadas a rho/fisiología , Neoplasias Esofágicas , MicroARNs/fisiología , Línea Celular Tumoral , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.
Asunto(s)
Células de Sertoli/metabolismo , Factor Esteroidogénico 1/metabolismo , Testículo/metabolismo , Animales , Animales Recién Nacidos , Hormona Antimülleriana/metabolismo , Apoptosis , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Genotipo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Noqueados , Fenotipo , Células de Sertoli/patología , Espermatogénesis , Factor Esteroidogénico 1/deficiencia , Factor Esteroidogénico 1/genética , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
To study the effects of gestational exposure to estrogen on early gonadal differentiation, pregnant mice were treated by sc injection of diethylstilbestrol (DES) or vehicle from embryonic day (E) 8.5 to E14.5, and gonads at E11.5, E12.5, and E14.5 were examined. Quantitative real-time RT-PCR and in situ hybridization revealed that mRNA levels of steroidogenic factor 1 (SF-1), a key regulator of gonadal differentiation, and several male gonad-specific genes, including Müllerian-inhibiting substance (MIS), steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein, were significantly decreased in the DES-treated testis, compared with the control testis at E12.5 and/or E14.5. Immunohistochemistry demonstrated that the staining intensities for SF-1 and MIS in Sertoli cells were apparently reduced in the DES-treated testis, compared with those of the controls, at E12.5 and E14.5. Because MIS, steroidogenic acute regulatory protein, cholesterol side-chain cleavage cytochrome P450, and Cerebellin 1 precursor protein are activated under the regulation of SF-1, the down-regulation of these factors may be due to reduced SF-1 expression. Immunohistochemistry for laminin-1 demonstrated that ovigerous cords in the DES-treated ovary were smaller than those in controls at E14.5. Moreover, the number of 5-bromo-2'deoxyuridine-5-monophosphate-labeled cells in the DES-treated testis was significantly reduced at E12.5 and E14.5, compared with controls, and that in the DES-treated ovary remained higher than that in the control ovary at E14.5. The results suggest that exogenous estrogens can alter sex-specific genetic pathways governing early differentiation and cell proliferation of both male and female gonads.
Asunto(s)
Dietilestilbestrol/toxicidad , Desarrollo Embrionario/efectos de los fármacos , Gónadas/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Animales , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , Proliferación Celular/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Gónadas/citología , Gónadas/embriología , Gónadas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Factor Esteroidogénico 1/genéticaRESUMEN
We established a mesenchymal stem cell clone, 5F9A, from rat bone marrow substrate adherent cells by repeated limiting dilutions. The cells have a fibroblastic shape and form intimate contacts with adjacent cells with interdigitations and junctions similar to adherence and tight junctions in a semi-confluent culture. Analysis of the phenotypes of these cells by RT-PCR and FACS demonstrated that they resembled mesenchymal stem cells, and the cells could differentiate into adiopocytes and osteoblasts under appropriate conditions in vitro showing their oligopotency. Furthermore, the cells were induced to become multinuclear cells by TPA (12-o-tetradecanoylphorbol 13-acetate) stimulation.
Asunto(s)
Adipocitos/ultraestructura , Diferenciación Celular/fisiología , Núcleo Celular/ultraestructura , Células Madre Mesenquimatosas/fisiología , Osteoblastos/ultraestructura , Animales , Línea Celular , Uniones Intercelulares/ultraestructura , Células Madre Mesenquimatosas/ultraestructura , Fenotipo , Ésteres del Forbol , RatasRESUMEN
The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.
Asunto(s)
División del Núcleo Celular/efectos de los fármacos , Citocinesis/efectos de los fármacos , Células Gigantes/citología , Células Gigantes/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Fusión Celular , Células Clonales , ADN/análisis , Genoma , Citometría de Barrido por Láser , Proteína Quinasa C/metabolismo , RatasRESUMEN
Certain hollow organs are known to form cysts when heterologously transplanted. In order to examine the usefulness of the phenomenon for regenerative medicine, rat urinary bladders and other organs were allo-transplanted under the subcutaneous tissue of the back. These transplanted tissues very often formed cysts covered with epithelia. The epithelia covered an area about twice the original size. In the case of the urinary bladder, the epithelium started moving from the edge of the transplants around day 3 after the operation, and as time proceeded, the tela submucosa and tunica muscularis also moved to encircle the epithelium, and formed the wall of the cyst. The basal laminae were formed under the newly expanded epithelium slightly behind the leading tip. All of the organs tested had the capability of cyst formation and epithelialization, although their rate differed between organs. The results are discussed with reference to the potential use of cyst formation for regenerating damaged organs.
Asunto(s)
Trasplante de Órganos/métodos , Regeneración/fisiología , Vejiga Urinaria/fisiología , Vejiga Urinaria/trasplante , Animales , Quistes/fisiopatología , Fenómenos Fisiológicos del Sistema Digestivo , Procedimientos Quirúrgicos del Sistema Digestivo , Epitelio/fisiología , Ratas , Ratas Wistar , Fenómenos Fisiológicos de la Piel , Tejido Subcutáneo , Tráquea/fisiología , Tráquea/trasplanteRESUMEN
Skins and hollow organs have been shown to form epithelialized cysts when transplanted into subcutaneous tissue of a recipient animal, expanding their surface areas. This system seems to offer a good potential for regenerating organs. We investigated the functional and structural contribution of epithelia and connective tissue compartments in this regeneration system with two experimental systems.
RESUMEN
Whole body gamma-ray irradiation of rats with caesium-137 (137Cs) at embryonic day 20 induced marked reduction of the weight of the testis. Body weight and other tissues, however, seemed to remain normal. By light microscopy, complete loss of germ cells was observed in the testis. Other components, such as Sertoli cells and interstitial cells, seemed to be normal. The testes from day 8 postpartum rats contained very few spermatogonia compared with newborn rats, indicating loss of germ cells between days 0 and 8. In the adult, 137Cs-irradiated testes showed two conspicuous features other than the loss of germ cells: empty vacuolar spaces between Sertoli cells and multilayered seminiferous tubule basal laminae (lamina densa). The junctional structures (ectoplasmic specializations) between Sertoli cells, however, seemed normal. The thickness of each layer of multilayered basal laminae was the same as that of normal rats and electron-lucent layers similar to lamina lucida were interposed between them. Of the empty vacuolar spaces between Sertoli cells, basal laminae bridge the gap. The basal laminae contained laminin, type IV collagen and heparan sulphate proteoglycan evenly distributed among layers, suggesting a normal composition. Rough estimation of the amount of basal laminae deposited in 137Cs-irradiated rats indicates that it is within a range similar to that in normal testis. These features imply that Sertoli cells are, in part, determined perinatally to produce basal laminae for germ-line cells.