RESUMEN
PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin.
Asunto(s)
ARN Largo no Codificante/genética , Factor de Unión a CCCTC , Estudios de Casos y Controles , Ciclo Celular/genética , Diferenciación Celular , Línea Celular , Proliferación Celular , Simulación por Computador , Epidermis/metabolismo , Epidermis/patología , Regulación de la Expresión Génica/efectos de la radiación , Silenciador del Gen/efectos de la radiación , Humanos , Inmunohistoquímica , Espacio Intracelular/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas Nucleares , Nucleofosmina , Unión Proteica , Transporte de Proteínas , Psoriasis/genética , ARN Largo no Codificante/metabolismo , Proteínas Represoras/metabolismo , Rayos UltravioletaRESUMEN
Psoriasis is a chronic inflammatory skin disease that affects approximately 2-4% of the population. We recently described a novel non-coding RNA, psoriasis susceptibility related RNA gene induced by stress (PRINS), that was overexpressed in non-lesional psoriatic epidermis, and its expression was induced by various stress factors such as serum starvation, contact inhibition, ultraviolet (UV)-B irradiation, viral infection and translational inhibition in HaCaT cells. In the present work we set out to compare the stress and microbial agent-induced PRINS expression in normal human keratinocytes (NHKs) and HaCaT cells. Since nuclear factor-κB (NF-κB) is involved in the cellular stress response, we sought to explore whether there is a connection between the NF-κB and PRINS-mediated signal transduction pathways in NHKs and HaCaT cells. We found that the PRINS expression responded differentially to various stress signals and microbial agents in HaCaT cells and in NHKs: after translational inhibition and UV-B treatment, similar induction of PRINS expression occurred with different time courses while after microbial agent treatment, the PRINS expression was significantly induced in HaCaT cells, whereas we could not detect similar changes in NHKs. To explore whether the known NF-κB abnormalities in HaCaT cells could be related to this differential PRINS expression, we silenced the PRINS gene expression with small interfering RNA (siRNA) in both HaCaT cells and in NHKs and monitored NF-κB signal transduction after lipopolysaccharide (LPS) treatment. Silencing of PRINS had no effect on LPS-induced NF-κB activity either in HaCaT cells or in NHKs. Our results indicate that PRINS probably affects keratinocytes functions independently of NF-κB signalling.
Asunto(s)
Queratinocitos/metabolismo , Psoriasis/metabolismo , ARN no Traducido/metabolismo , Antígenos Bacterianos/metabolismo , Línea Celular , Cicloheximida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Queratinocitos/efectos de la radiación , FN-kappa B/genética , FN-kappa B/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Psoriasis/genética , ARN Interferente Pequeño/genética , ARN no Traducido/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Numerous epidemiological studies have proposed a link between herpes simplex virus (HSV) infection and several common chronic neuropsychiatric and neurodegenerative diseases. Experimental HSV infection of mice can lead to chronic behavioral and neurological deficits and chronic pain. While neuron injury and loss are well-documented consequences of the acute phase of infection, the pathologic consequences of latent HSV infection are poorly understood. To determine whether latent HSV infection can cause neuronal injury in mice, trigeminal ganglia (TG) derived from adult BALB/c mice 1, 12 and 31 weeks after corneal HSV type 1 (HSV-1) inoculation were analyzed for evidence of productive or latent HSV-1 infection, inflammation and changes in neuron size, density and number. We found that latent HSV-1 infection between 12 and 31 weeks after corneal virus inoculation was associated with inflammation and progressive deficits in mean neuron diameter, neuronal nucleus diameter, neuron density and neuron number in the TG relative to mock-infected controls. The extent of neuronal injury during latent infection correlated with the extent of inflammation. These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases.
Asunto(s)
Infecciones por VIH/patología , Herpesvirus Humano 1/patogenicidad , Neuronas/patología , Ganglio del Trigémino/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Edad , Análisis de Varianza , Animales , Recuento de Células/métodos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Viral de la Expresión Génica , Inflamación/etiología , Inflamación/virología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Neuronas/virología , Fosfoproteínas/metabolismo , Factores de Tiempo , Ganglio del Trigémino/virología , Proteínas Virales/metabolismoRESUMEN
In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.
Asunto(s)
Desdiferenciación Celular/fisiología , Células Epidérmicas , Melanocitos/fisiología , Adulto , Desdiferenciación Celular/genética , Células Cultivadas , Toxina del Cólera/farmacología , Medios de Cultivo Condicionados/farmacología , Dendritas/efectos de los fármacos , Dendritas/fisiología , Epidermis/metabolismo , Epidermis/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Queratinocitos/metabolismo , Queratinocitos/fisiología , Queratinocitos/efectos de la radiación , Melanocitos/citología , Melanocitos/metabolismo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/genética , Pigmentación de la Piel/fisiología , Acetato de Tetradecanoilforbol/farmacología , Rayos UltravioletaRESUMEN
Herpes simplex virus type 1 (HSV-1) is an important human pathogen and a leading cause of infectious blindness in the developed world. HSV-1 exploits heparan sulfate proteoglycans (HSPG) for attachment to cells. While the significance of heparan sulphate (HS) moieties in HSV-1 infection is well established, the role of specific proteoglycan core proteins in the infection process remains poorly understood. The objective of this study was to assess the roles of syndecan-1 and syndecan-2 core proteins in HSV-1 infection, both of which are expressed by many HSV-1 target cell types. Our results demonstrate that syndecan-1 and syndecan-2 gene silencing by RNA interference reduces HSV-1 entry, plaque formation and facilitates cell survival. Furthermore, HSV-1 infection increases syndecan-1 and syndecan-2 protein synthesis and a resultant increase in cell surface expression of HS. Our observations suggest that changes in syndecan-1 and syndecan-2 expression levels may be related to active viral infection. Taken together, our findings provide new insights into HSPG functions during HSV-1 entry and spread.
Asunto(s)
Herpesvirus Humano 1/patogenicidad , Interacciones Huésped-Patógeno , Sindecano-1/metabolismo , Sindecano-2/metabolismo , Internalización del Virus , Animales , Línea Celular , Chlorocebus aethiops , Silenciador del Gen , Humanos , Interferencia de ARN , Sindecano-1/antagonistas & inhibidores , Sindecano-2/antagonistas & inhibidoresRESUMEN
PURPOSE: Herpes virus entry mediator (HVEM) plays a critical role in the regulation of inflammation through interaction with its natural ligands LIGHT and lymphotoxin alpha and also serves as one of the entry receptors of herpes simplex virus (HSV). The purpose of this study was to better understand the expression of HVEM in the cornea and trigeminal ganglia (TG), which are important targets of HSV infection. MATERIALS AND METHODS: Immunohistochemistry was used to define HVEM expression in the cornea and TG of normal and HSV-1 infected mice euthanized 2 to 5 days or 7 months following corneal inoculation of virus. RESULTS: We found that HVEM is widely expressed in the normal corneal epithelium and endothelium, is weakly and focally expressed in the corneal stroma, and is expressed in a portion of neurons and non-neuronal cells in the TG. Acute HSV-1 keratitis and ganglionitis were associated with increased HVEM expression in the corneal epithelium and stroma and in neurons and non-neuronal cells of TG, and many inflammatory cells in these tissues also expressed HVEM. TG derived from mice 7 months after virus inoculation demonstrated latent HSV-1 infection that was associated with increased HVEM expression in neurons and non-neuronal cells relative to uninfected control tissues. Latent TG also contained focal infiltrates of mononuclear inflammatory cells, many of which expressed HVEM. Corneas derived from latently infected mice demonstrated chronic keratitis, with no evidence of virus replication or increased HVEM expression in the corneal epithelium, and inflammatory cells present showed only weak HVEM expression. CONCLUSIONS: HVEM is expressed in the cornea and TG and therefore may serve as an HSV entry receptor in these tissues. Furthermore, these findings raise the possibility that changes in HVEM expression following ocular HSV-1 infection can modulate HSV spread and infection-induced inflammation in the cornea and TG.