RESUMEN
Various actions trigger pleasure (reward) or aversion (punishment) as emotional responses. Emotional factors that negatively affect brain neural control processes for long periods of time might cause various mental diseases by inducing neuronal changes. In the present study, newly developed PC12m12 cells which areãhighly sensitivity to neurotransmitters such as acetylcholine (ACh), were used. Exposing the cells to plasma from rats that had been subjected to intracranial self-stimulation (ICSS) markedly upregulated neurite outgrowth. In addition, voluntary running in a wheel or forced on a rotating rod was used to induce behavioral excitation in rats, and examinations of their plasma confirmed that the ICSS-induced neurite outgrowth was not associated with the ICSS behavior itself. Furthermore, immunoblotting and treatment with U0126, an ERK (extracellular signal-regulated kinase) antagonist, showed that the ICSS-induced neurite outgrowth was related to neuronal ERK activity. Exposing the same cells to plasma from rats that had been subjected to immobilization (IMM) also increased neurite outgrowth. Although the degree of enhancement was not as great as that seen after the ICSS rat plasma treatment, it was less than that observed after treatment with ACh as a positive control. These results indicate that ICSS or IMM lead to varying degrees of morphological changes, such as enhanced neurite outgrowth, in PC12m12 cells, but the neuronal signal transduction pathways underlying these effects differ; i.e.,the former morphological change might involve the activation of the ERK pathway, whereas the latter changes might not. Using PC12m12 cells which exhibit sensitivity to neurotransmitters, it might be possible to clarify the pathogeneses of mental diseases at the neuronal level and search for therapeutic drugs.
Asunto(s)
Conducta Animal/fisiología , Emociones/fisiología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Crecimiento Nervioso/fisiología , Neuritas/fisiología , Neurogénesis/fisiología , Recompensa , Animales , Conducta Animal/efectos de los fármacos , Butadienos/farmacología , Emociones/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Factor de Crecimiento Nervioso/efectos de los fármacos , Neuritas/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Nitrilos/farmacología , Células PC12 , Placer/fisiología , Ratas , Ratas WistarRESUMEN
We investigated the differentiation and activation of p38 MAPK induced by contrast bath in drug-hypersensitive PC12m3 mutant cells. The rate of neurite outgrowth in PC12m3 cells induced by contrast bath was much higher than that induced by warming or cooling alone or that induced by two warmings with an interval of room temperature, indicating that contrast bath has a synergistic effect. The results of an experiment using a p38 MAPK inhibitor, SB203580, showed that neurite outgrowth of PC12m3 cells induced by contrast bath is p38 MAPK-dependent. Moreover, p38 MAPK activity induced by contrast bath was greater than that induced by warming or cooling alone, indicating that the synergistic effect of a contrast bath on neurite outgrowth depends on the activity of p38 MAPK. Since calcium ions are involved in the activations of P38 MAPK, we investigated the effect of the TRP ion channel inhibitor (Capsazepine) that inhibits calcium influx in the cells. Neurite outgrowth induced by contrast bath treatment was greatly suppressed by the addition of Capsazepine. These findings suggest that calcium dependent activation of the p38 MAPK pathway induced by contrast bath is responsible for the neurite outgrowth of PC12m3 cells.
RESUMEN
Insulin interacts with the insulin receptor, and the activated receptor promotes activity of the phosphoinositide-3 kinase (PI3K) enzyme. A decrease in insulin or insulin-like growth factor 1 (IGF-1) signaling increases the lifespan in mammalian species. We found that a point mutation in the C-SH2 domain of the p85ß regulatory subunit of PI3K results in a prolonged lifespan. In p85ß mutant cells, nerve growth factor (NGF) activates the longevity protein FOXO, and the mutant p85ß gene produces strong resistance to oxidative stress, which contributes to aging. The p85ß gene mutation causes increased serum insulin and low blood glucose in p85ß mutant transgenic mice. Our results indicate that the p85ß mutant allele alters the activity of downstream targets of PI3K by NGF and platelet-derived growth factor (PDGF) but not by insulin. We report that a point mutation in the C-SH2 domain of p85ß transforms p85ß into a novel anti-aging gene by abnormally regulating PI3K.
Asunto(s)
Envejecimiento , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Animales , Glucemia/análisis , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Crecimiento Nervioso/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Factor de Crecimiento Derivado de Plaquetas/farmacología , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Dominios Homologos src/genéticaRESUMEN
We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in electrical stimulation-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to electrical stimulation at 100 mA for 30 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The frequency of neurite outgrowth induced by electrical stimulation was approximately 10-fold greater than that of neurite outgrowth induced by NGF alone. The neurite outgrowth induced by electrical stimulation was inhibited by a specific p38 MAPK inhibitor, SB203580. The activation of p38 MAPK induces activation of the transcription factor CREB. The activation of CREB induced by electrical stimulation was inhibited by SB203580. Longer electrical stimulation of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that electrical stimulation-induced activation of the p38 MAPK/CREB pathway is responsible for the neurite outgrowth and survival of PC12m3 cells.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Neuritas/metabolismo , Animales , Estimulación Eléctrica , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuritas/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Células PC12/efectos de los fármacos , Piridinas/farmacología , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIM: In mammals, rewarding and aversive states are motivational drivers of behavioral expression. However, it is unclear whether such states affect neuronal functions at the level of individual neurons. In the present study, the neuronal effects of rewarding and aversive states were investigated in using PC12 mutant cells (PC12m3 cells) with low sensitivity to nerve growth factor. MAIN METHODS: The intracranial self-stimulation (ICSS) and immobilization (IMM) methods were used to create rewarding and aversive states, respectively, in rats. Furthermore, experiments involving voluntary running on a wheel and forced running on a rotating rod were used to evaluate the effects of behavioral excitement on neurons. Then, the effects of plasma samples collected from the animals on neurite extension were examined microscopically, and p38 mitogen-activated protein kinase (MAPK) activity was assessed using Western blotting. KEY FINDINGS: Plasma samples from the ICSS and IMM rats facilitated neurite outgrowth to different degrees. However, their effects were not influenced by behavioral excitement. Furthermore, the plasma from the ICSS rats also induced upregulated p38 MAPK activity, whereas that from the IMM rats produced the same or slightly lower levels of MAPK activity to the control plasma. SIGNIFICANCE: These findings indicate that rewarding and aversive states might cause morphological changes, such as neurite extension. As for the effects of these states on p38 MAPK activity, the former state might directly increase p38 MAPK activity, but the latter state might have no effect or cause a slight reduction in p38 MAPK activity.
Asunto(s)
Inmovilización/psicología , Neuritas/metabolismo , Recompensa , Autoestimulación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Reacción de Prevención/fisiología , Conducta Animal , Western Blotting , Masculino , Factor de Crecimiento Nervioso/metabolismo , Células PC12 , Ratas , Ratas Wistar , Carrera/fisiología , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Factors that trigger emotional expression may be divided into two patterns according to the type of motivation, acquiring reward (pleasure) and avoiding aversion (punishment). Repeated exposure to certain external stimuli accompanied by aberrant motivation may produce psychiatric diseases such as bipolar disorder and addiction via dysregulation of the central nervous system. However, neurobiological underpinnings of such diseases have not been clarified, especially at the neuronal level. In the present study, plasma from rats undergoing intracranial self-stimulation (ICSS) produced neurite outgrowth in PC12-variant cells (PC12m3). Stimulated PC12m3 cells also exhibited heightened activity of the p38 MAPK pathway. These findings indicate that reward states lead to not only morphological changes but also increases in p38 MAPK activity at the neuronal level in the central nervous system.
Asunto(s)
Haz Prosencefálico Medial/fisiología , Neuritas/fisiología , Recompensa , Autoestimulación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Estimulación Eléctrica , Masculino , Neuritas/metabolismo , Células PC12 , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
We examined the effect of 1α-hydroxyvitamin D3 [1α(OH)D3] on mice in the forced swimming test. Intragastric administration of 1.0 µg/kg of 1α(OH)D3 reduced immobility time in the forced swimming test. At all concentrations tested (0.5, 1.0, 2.0 µg/kg), 1α(OH)D3 had no effect on locomotor activity, compared with controls. These results suggest that 1α(OH)D3 may have antidepressant-like activity.
Asunto(s)
Antidepresivos , Hidroxicolecalciferoles/farmacología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos ICR , NataciónRESUMEN
PURPOSE: To investigate whether a steam foot spa improves cognitive impairment in geriatric inpatients. METHODS: Geriatric inpatients with cognitive impairment were given a steam foot spa treatment at 42°C for 20 minutes for 2 weeks (5 days/week). Physiological indicators such as blood pressure, percutaneous oxygen saturation, pulse, tympanic temperature, and sleep time and efficiency were assessed. Cognitive function and behavioral and psychological symptoms of dementia were assessed using the Mini-Mental State Examination, Dementia Mood Assessment Scale, and Dementia Behavior Disturbance scale. RESULTS: Significant decreases in systolic (P < 0.01) and diastolic blood pressure (P < 0.05) along with a significant increase in tympanic temperature (P < 0.01) were observed after the steam foot spas. A significant improvement was seen in the Mini-Mental State Examination score (P < 0.01) and the overall dementia severity items in Dementia Mood Assessment Scale (P < 0.05). LIMITATIONS: Japanese people are very fond of foot baths. However, it is difficult to understand why inpatients cannot receive steam foot baths. In this study, a control group was not used. Raters and enforcers were not blinded. CONCLUSION: The results of this pilot study suggest that steam foot spas mitigate cognitive impairment in geriatric inpatients.
Asunto(s)
Trastornos del Conocimiento/terapia , Pie , Hidroterapia/métodos , Anciano , Presión Sanguínea , Temperatura Corporal , Femenino , Humanos , Japón , Masculino , Pruebas Neuropsicológicas , Consumo de Oxígeno/fisiología , Proyectos Piloto , Pulso Arterial , Sueño/fisiología , Estadísticas no Paramétricas , Vapor , Resultado del TratamientoRESUMEN
We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.
Asunto(s)
Ciclohexenos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células PC12/efectos de los fármacos , Aceites de Plantas/farmacología , Terpenos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Animales , Ciclohexenos/química , Limoneno , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/fisiología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neurogénesis , Ratas , Transducción de Señal , Terpenos/químicaRESUMEN
We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. Elongation and re-elongation of the root during the recovery process from Al injury occurred only in the apical 5-mm region of the pea root. With the model system of the pea root for recovery from Al injury, images of the root characterized by zonal staining with Evans blue showed the existence of two regions in the root apex consisting of rupture and zonary stained regions. Ruptures enlarged by increase in their depth but without widening of the intervals among zonary stained regions in the roots treated with Al continuously. On the other hand, intervals of the zonary stained regions were widened due to re-elongation of the root and were narrow in the rupture region in the recovery root.
Asunto(s)
Aluminio/toxicidad , Azul de Evans/metabolismo , Meristema/efectos de los fármacos , Meristema/crecimiento & desarrollo , Pisum sativum/efectos de los fármacos , Pisum sativum/crecimiento & desarrollo , Coloración y Etiquetado , Muerte Celular/efectos de los fármacos , Meristema/anatomía & histología , Meristema/citologíaRESUMEN
Among the 3 mitogen-activated protein kinases--ERK, p38 MAPK and JNK--JNK has been suggested to participate in apoptosis, whereas p38 MAPK is thought to be part of the differentiation response. There are many common inducers of JNK and p38 MAPK, but the mechanisms underlying the differential response to apoptosis and differentiation are poorly understood. We found that heatshock activated p38 MAPK at 3 min after exposure to a temperature of 44 degrees C in stress-hypersensitive PC12m3 mutant cells, while it activated JNK at 20 min after the same heat treatment. However, heatshock activated p38 MAPK 5min after heat treatment and JNK 10 min after heat treatment in PC12 parental cells. The extent of phosphorylation of p38 MAPK induced by heat shock in PC12m3 cells was significantly greater than that in PC12 parental cells, and a high level of heat-shock-induced neurite outgrowth was observed only in PC12m3 cells. On the other hand, heat-shock-induced JNK activation appeared more quickly and apoptosis started earlier in PC12 parental cells. These findings indicate that short stress induces p38 MAPK and longer stress induces JNK, and that the response of these kinases to heat shock differs depending on cell type.
Asunto(s)
Apoptosis/fisiología , Respuesta al Choque Térmico/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuritas/fisiología , Neuronas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Diferenciación Celular/fisiología , Neuronas/ultraestructura , Células PC12 , Ratas , Estrés Fisiológico/fisiologíaRESUMEN
The aim of this study was to determine the optimal heat treatment conditions for enhancement of pressed silk-mediated 3D-like proliferation of normal human dermal fibroblasts, as well as to determine the responses to heat shock of cells and intracellular signaling pathways. The beginning of 3D-like pattern formation of cells was observed in the second week after the start of the experiment. The mean rates of beginning of 3D-like pattern formation by cells heat-treated at 40 masculineC and 43 masculineC for 10 min were significantly higher (3.2- and 8.6-fold, respectively) than that of untreated cells. We found that apoptosis had occurred in 7.5% and 50.0% of the cells at one week after heat treatment for 10 min at 43 masculineC and 45 masculineC, respectively. Western blot analysis demonstrated that phosphorylation of p38 MAPK and that of Hsp27 were markedly increased by heat treatment at 43 masculineC for 10 min. The results of an experiment using a p38 MAPK inhibitor and Hsp27 inhibitor suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation and that Hsp27 contributes to the inhibition of apoptosis. The results of this study should be useful for further studies aimed at elucidation of the physiologic mechanisms underlying thermotherapy.
Asunto(s)
Técnicas de Cultivo de Célula , Fibroblastos/citología , Calor , Seda/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Masculino , Fosforilación/efectos de los fármacos , Seda/farmacología , Temperatura , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Fenilpropionatos/farmacología , Própolis/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Alcaloides/química , Alcaloides/metabolismo , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Apoptosis/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Humanos , Ratones , Estructura Molecular , Neuritas/ultraestructura , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Células PC12/citología , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Fenilpropionatos/química , Própolis/farmacología , Piridonas/química , Piridonas/metabolismo , RatasRESUMEN
The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de la radiación , Microondas/efectos adversos , Neuritas/enzimología , Neuritas/efectos de la radiación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Teléfono Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Respuesta al Choque Térmico/fisiología , Imidazoles/farmacología , Células PC12 , Piridinas/farmacología , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The aim of the present study was to determine both the minimal and the optimal conditions under which heat treatment is effective for enhancing 3D (three-dimensional)-like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or 45 degrees C for 2, 10, 15, 20, 30, 45, 60, 90, 180 and 360 min. Then minimal and optimal conditions of temperature and duration of heat treatment for induction of 3D-like proliferation of cells were determined. The minimal conditions of heat treatment to induce 3D-like proliferation were 43 degrees C for 2 min and the optimal conditions were 43 degrees C for 10 min. The mean rates of formation of 3D-like proliferation patterns by cells heat-treated at 43 degrees C for 2 and 10 min were significantly higher (1.7- and 3.7-fold respectively) than that by untreated cells (P<0.05). We also observed significantly greater effects of heat treatment on 3D-like proliferation at 40 degrees C for 90 or 180 min and at 41.5 degrees C for 15 min and 44 degrees C for 10 min. We found that apoptosis had occurred in 7.5 and 87.0% of the cells at 1 week after heat treatment at 43 degrees C for 10 min and 30 min respectively. Western-blot analysis demonstrated that phosphorylation of p38 MAPK (mitogen-activated protein kinase) was markedly increased by heat treatment at 43 degrees C for 10 min. These findings suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation. The results of the present study should be useful for further studies aimed at elucidation of the physiological mechanisms underlying thermotherapy and hyperthermia.
Asunto(s)
Proliferación Celular , Durapatita , Fibroblastos/citología , Respuesta al Choque Térmico , Calor , Animales , Apoptosis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C3H , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used in conjunction with the antibody to the His-tags to determine the topology of ALMT1. This study shows that the ALMT1 protein contains six transmembrane domains with the amino and carboxyl termini located on the extracellular side of the plasma membrane.
RESUMEN
The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada/citología , Transformación Celular Viral , Fibroblastos/citología , Ligamento Periodontal/citología , Telomerasa/genética , Transfección , Adulto , Antígenos Transformadores de Poliomavirus/metabolismo , Calcificación Fisiológica/genética , Línea Celular Transformada/metabolismo , Proliferación Celular , Supervivencia Celular , Fibroblastos/metabolismo , Fibroblastos/virología , Humanos , Masculino , Ligamento Periodontal/metabolismo , Telomerasa/metabolismoRESUMEN
Regulation of the biocompatibility of compositional hydroxyapatite (HA) with cells is affected by various environmental factors. The aim of this study was to determine whether the p38 mitogen-activated protein kinase (MAPK) pathway has a key role in enhancement of HA-mediated three-dimensional (3D)-like proliferation of mouse fibroblasts after heat treatment. C3H10T1/2 mouse fibroblasts were cultured with HA granules for 10 weeks after heat treatment at 44 degrees C for 5, 10, 20, and 30 min. The mean rate of formation of 3D-like proliferation patterns by cells heat treated for 20 min was only 2.1-fold higher than that by untreated cells, but the mean rates of formation of 3D-like proliferation patterns by cells heat treated for 5 and 10 min were significantly higher (3.7- and 3.3-fold higher, respectively) than that by untreated cells (p < 0.01). Western blot analysis demonstrated that phosphorylation of p38 MAPK was markedly increased by heat treatment at 44 degrees C for 5 and 10 min. In addition, the activation of heat shock-induced p38 MAPK was markedly reduced by treatment at 44 degrees C for 30 min. We concluded that 3D-like proliferation of heat-treated cells was induced by activation of p38 MAPK. The results of this study should be useful for further studies aimed at elucidation of regulation of the biocompatibility of compositional HA with cells.
Asunto(s)
Proliferación Celular , Durapatita , Fibroblastos/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Calor , Ratones , Ratones Endogámicos C3HRESUMEN
We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.
Asunto(s)
Calor , Neuritas/efectos de la radiación , Choque , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting/métodos , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Células PC12 , Piridinas/farmacología , Ratas , Factores de TiempoRESUMEN
Various undifferentiated embryonic stem (ES) cells can grow on mouse embryonic fibroblast (MEF) feeders. However, the risk of zoonosis from animal feeders to human ES cells generally excludes the clinical use of these human ES cells. We have found that human placenta is a useful source of feeder cells for the undifferentiated growth of primate ES cells. As on MEF feeders, primate ES cells cultured on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) cells had undifferentiated growth. The cultured primate ES cells expressed Oct-4, alkaline phosphatase, and SSEA-4. The primate ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into severe combined immunodeficient mice. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications.