RESUMEN
The aim of the present study was to evaluate the regulation of membrane-anchored mucin MUC16 by proinflammatory cytokines and bacterial components at the ocular surface. Expression and distribution of MUC16 in conjunctival (HCjE) and corneal (HCE) epithelial cell lines was monitored by RT-PCR and immunohistochemistry. To determine the regulation of MUC16, cultured HCjEs and HCEs were stimulated with different cytokines, bacterial components and bacterial supernatants, and analyzed by real-time PCR, immunodot blot and immunohistochemistry. The results indicate that MUC16 is differentially regulated between HCjEs and HCEs after challenge with inflammatory mediators and suggest shedding of MUC16 from the ocular surface epithelia into the tear film. This seems to be precisely regulated. MUC16 shedding can be differentially increased and decreased, suggesting a protective function of membrane-anchored MUC16 and supporting the hypothesis that dysregulation of membrane-anchored MUC16 at the ocular surface may be involved in dry eye pathology.
Asunto(s)
Antígeno Ca-125/genética , Conjuntiva/citología , Córnea/citología , Células Epiteliales/fisiología , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Línea Celular , Conjuntiva/fisiología , Córnea/fisiología , ADN Complementario/genética , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Inflamación/fisiopatología , Interferón gamma/farmacología , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Peptidoglicano/farmacología , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Proteínas de Anclaje a la Quinasa A/metabolismo , Sitios de Unión , Unión Competitiva , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Piridinas/síntesis química , Piridinas/metabolismoRESUMEN
The second messenger cyclic adenosine monophosphate (cAMP), which is produced by adenylyl cyclases following stimulation of G-protein-coupled receptors, exerts its effect mainly through the cAMP-dependent serine/threonine protein kinase A (PKA). Due to the ubiquitous nature of the cAMP/PKA system, PKA signaling pathways underlie strict spatial and temporal control to achieve specificity. A-kinase anchoring proteins (AKAPs) bind to the regulatory subunit dimer of the tetrameric PKA holoenzyme and thereby target PKA to defined cellular compartments in the vicinity of its substrates. AKAPs promote the termination of cAMP signals by recruiting phosphodiesterases and protein phosphatases, and the integration of signaling pathways by binding additional signaling proteins. AKAPs are a heterogeneous family of proteins that only display similarity within their PKA-binding domains, amphipathic helixes docking into a hydrophobic groove formed by the PKA regulatory subunit dimer. This review summarizes the current state of information on compartmentalized cAMP/PKA signaling with a major focus on structural aspects, evolution, diversity, and (patho)physiological functions of AKAPs and intends to outline newly emerging directions of the field, such as the elucidation of AKAP mutations and alterations of AKAP expression in human diseases, and the validation of AKAP-dependent protein-protein interactions as new drug targets. In addition, alternative PKA anchoring mechanisms employed by noncanonical AKAPs and PKA catalytic subunit-interacting proteins are illustrated.