RESUMEN
Transcription factor SOX4 has been implicated in skeletal myoblast differentiation through the regulation of Cald1 gene expression; however, the detailed molecular mechanism underlying this process is largely unknown. Here, we demonstrate that SOX4 acetylation at lysine 95 by KAT5 (also known as Tip60) is essential for Cald1 promoter activity at the onset of C2C12 myoblast differentiation. KAT5 chromodomain was found to facilitate SOX4 recruitment to the Cald1 promoter, which is involved in chromatin remodeling at the promoter. Chromatin occupancy analysis of SOX4, KAT5, and HDAC1 indicated that the expression of putative SOX4 target genes during C2C12 myoblast differentiation is specifically regulated by the molecular switching of the co-activator KAT5 and the co-repressor HDAC1 on SOX4 transcriptional activation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Histona Acetiltransferasas/genética , Histona Desacetilasa 1/genética , Mioblastos/metabolismo , Factores de Transcripción SOXC/genética , Transactivadores/genética , Acetilación , Secuencia de Aminoácidos , Animales , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Diferenciación Celular/genética , Línea Celular , Genes Reporteros , Células HEK293 , Histona Acetiltransferasas/metabolismo , Histona Desacetilasa 1/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Lisina Acetiltransferasa 5 , Ratones , Datos de Secuencia Molecular , Mioblastos/citología , Regiones Promotoras Genéticas , Factores de Transcripción SOXC/metabolismo , Alineación de Secuencia , Transducción de Señal , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: An effective treatment strategy for acne vulgaris is the reduction of Propionibacterium acnes in the skin. The Helicobacter pylori-derived synthetic antimicrobial peptide HPA3NT3 is a customized α-helical cationic peptide with antibacterial and anti-inflammatory activity. OBJECTIVES: To examine the role of HPA3NT3 as a treatment against P. acnes-induced skin inflammation. METHODS: Morphological alteration of individual P. acnes cells by HPA3NT3 was visualized by scanning electron microscopy. Modulation by HPA3NT3 of a number of P. acnes-induced innate immune responses was analysed in vitro using cultured normal human keratinocytes (HKs), and in vivo using the ICR mouse, a well-established model for P. acnes-induced skin inflammation. RESULTS: The minimum inhibitory concentration of HPA3NT3 against P. acnes was low (0·4 µmol L(-1)). HPA3NT3 showed no cytotoxicity to HK cells at the concentrations used in our in vitro and in vivo studies. Treatment with HPA3NT3 in vitro induced morphological disruptions in P. acnes cells suggestive of a bactericidal effect. HPA3NT3 significantly decreased P. acnes-induced interleukin-8 expression and intracellular calcium mobilization in HK cells by inhibiting P. acnes-activated Toll-like receptor 2-mediated nuclear factor-κB signalling pathways. Intradermal injection of HPA3NT3 in vivo effectively decreased viable P. acnes, as well as erythema, swelling and inflammatory-cell infiltration in ICR mouse ears inoculated with P. acnes. CONCLUSIONS: Our data suggest that HPA3NT3 has potential as a therapeutic agent for acne vulgaris due to its antimicrobial effects on P. acnes and its ability to block P. acnes-induced inflammation.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Helicobacter pylori , Fragmentos de Péptidos/farmacología , Proteínas Ribosómicas/farmacología , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Animales , Calcio/metabolismo , Células Cultivadas , Eritema/tratamiento farmacológico , Humanos , Inyecciones Intradérmicas , Interleucina-8/biosíntesis , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , FN-kappa B/metabolismo , Propionibacterium acnes/efectos de los fármacos , ARN Mensajero/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
PURPOSE: Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness. Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS). In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4). METHODS: CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry. LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization. Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA. The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry. RESULTS: Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14. LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines. CD14 mRNA expression in corneal epithelial cells was upregulated by LPS. In addition to CD14, corneal epithelial cells expressed the functional LPS receptor-signaling protein TLR4, which was also augmented by LPS. CONCLUSIONS: The cornea expresses functional CD14 and TLR4 LPS receptor proteins. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.