ABSTRACT
AIM: With regards to nanoparticles, all biomedical applications require cellular uptake, which to date remains a hurdle to further progress. This study aims to compare both the attractive force of a static magnetic field and the cell penetrating capability of penetratin; two techniques currently employed to enhance cell uptake. MATERIALS & METHODS: Fluorescent magnetic nanoparticles were functionalized with penetratin and cells were challenged with or without the particles in the presence/absence of a static magnetic field (350 mT). Following analysis of the magnetic field applied, cellular uptake and behavior was assessed in terms of fluorescence microscopy, clathrin and caveolin levels, scanning electron microscopy and transmission electron microscopy. RESULTS: Modeling of the field applied demonstrated varying field patterns across the cell culture area, reflected by higher particle uptake at higher field strengths. Both penetratin and the magnetic field increased cell uptake with penetratin proving more efficient. Interestingly, the magnetic field stimulated clathrin-mediated endocytosis and subsequent particle uptake.
Subject(s)
Carrier Proteins/pharmacokinetics , Ferric Compounds/pharmacokinetics , Fibroblasts/metabolism , Magnetic Fields , Metal Nanoparticles/administration & dosage , Carrier Proteins/chemistry , Caveolins/pharmacokinetics , Cell-Penetrating Peptides , Cells, Cultured , Clathrin/pharmacokinetics , Endocytosis/radiation effects , Ferric Compounds/chemistry , Ferric Compounds/radiation effects , Fibroblasts/ultrastructure , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Microscopy, Electron/methods , Particle SizeABSTRACT
Las caveolas participan en múltiples procesos celulares tales como el transportevesicular, homeostasis del colesterol, regulación de la señalización intracelular,por integrinas y proliferación celular. Sin embargo, su función en el hígado no estábien establecida. La expresión de caveolina 1 (Cav), la proteína más abundante enlas caveolas, está bien descrita en el hígado y en varias líneas de hepatocitos y enhígado cirrótico humano y en carcinoma hepatocelular. Sin embargo, el papel deCav-1 en la fisiopatología hepática es controvertido, ya que se ha propuesto un papel crítico en el proceso de regeneración tras hepatectomía parcial (HP). Contrariamentea esta observación, nuestros datos sugieren que Cav-1 aumenta en elhígado regenerante, con una re-distribución de la proteína desde las caveolas haciadominios no caveolares. Además, la Cav-1 localizada en estas fracciones está fosforiladaen la tirosina 14. A pesar de ello, el gen de la Cav-1 es dispensable parala regeneración hepática tras HP, tal como se deduce de animales que carecen deeste gen. En conjunto, estos datos muestran un papel dinámico de la Cav-1 en laproliferación hepática tras HP y en líneas hepáticas en cultivo, pero con mínimasimplicaciones en el proceso regenerativo
Although caveolae participate in many cellular processes such as vesicular transport,cholesterol homeostasis, regulation of signal transduction, integrin signalingand cell growth, their role in liver remains elusive. Expression of caveolin 1 (Cav),the most abundant protein of caveolae, has been reported in liver and in differenthepatocyte cell lines, in human cirrhotic liver and in hepatocellular carcinomas.However, the role of Cav-1 in liver pathophysiology remains controversial and acritical role in regeneration after partial hepatectomy (PH) has been reported.Opposite to this observation, our data support the view that Cav-1 increases inliver after PH with a redistribution of the protein from the caveolae enricheddomain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolarfraction is phosphorylated in tyrosine 14 (Tyr14). Even though, the Cav-1 geneis dispensable for liver regeneration after PH as deduced from data obtained withcommercially available animals lacking this gene. Taken together these resultssupport a dynamic role for Cav-1 in liver proliferation both in vivo after PH, andin vitro in cultured hepatic cell lines, but with minimal implications in the liverregeneration process
Subject(s)
Caveolins/chemistry , Caveolins/pharmacology , Liver Regeneration , Liver/chemistry , Hepatectomy/methods , Hepatectomy/rehabilitation , Caveolins/analysis , Caveolins/chemical synthesis , Caveolins/pharmacokinetics , Liver Regeneration/immunology , Liver Regeneration/physiology , Caveolae/chemistry , Caveolae , Liver , Hepatocyte Growth Factor/chemical synthesis , Hepatocyte Growth Factor/pharmacologyABSTRACT
Introducción. La activación o la sobreexpresión del receptor activado por proliferadores peroxisómicos * (PPAR*) induce la expresión de caveolina-1 en diferentes tipos celulares. El objetivo de este estudio ha sido evaluar si los agonistas PPAR regulan la expresión de la caveolina-1 en macrófagos, y explorar los posibles mecanismos implicados. Material y métodos. Se diferenciaron monocitos THP-1 por exposición a PMA durante 24 h y, posteriormente, se trataron con rosiglitazona a diferentes dosis y durante distintos períodos. Los valores de ARNm se determinaron por la reacción de la transcriptasa inversa acoplada a la reacción en cadena de la polimerasa (RT-PCR), y la expresión de la proteína mediante western-blot. Resultados. El tratamiento con la rosiglitazona aumentó los valores de ARNm y de proteína de caveolina-1 de forma dependiente de la dosis y el tiempo en macrófagos THP-1. Esta inducción no se observó en presencia de inhibidores de la transcripción o de la síntesis de novo de proteínas. El incremento de la expresión de caveolina-1 producido por rosiglitazona no se relaciona con el estado de diferenciación celular y parece ser dependiente de la activación PPAR, ya que la presencia del antagonista PPAR GW9662 lo anuló por completo. Por último, se ha identificado un elemento de respuesta a proliferadores peroxisómicos (PPRE) funcional en el promotor del gen de la caveolina-1, que se activa por el tratamiento con rosiglitazona en macrófagos THP-1. Conclusiones. La rosiglitazona incrementa la expresión de caveolina-1 en macrófagos, a través de la activación de los PPAR y, probablemente, como consecuencia de la unión al PPRE identificado en la secuencia del promotor del gen de la caveolina-1 (AU)
Introduction. Peroxisome proliferator-activated receptor * (PPAR*) activation or overexpression induce caveolin-1 expression in several cell types. The aim of this study was to ascertain whether PPAR agonists could also regulate the caveolin-1 gene in macrophages, and to investigate the mechanisms involved. Materials and methods. PMA-treated THP-1 monocytes were incubated with rosiglitazone at different concentrations and for different periods of time. MRNA levels were determined by RT-PCR and protein expression by Western blot. Results. Our experiments demonstrated that rosiglitazone dose- and time-dependently increased caveolin-1 mRNA and protein in THP-1 macrophages. This induction was not observed in the presence of transcription inhibitors or de novo protein synthesis. We also showed that the increase in caveolin-1 elicited by rosiglitazone was not related to macrophage differentiation. This inductive effect seems to be dependent on PPAR activation, since the PPAR antagonist GW9662 abolished it. Finally, we identified a functional peroxisome proliferator response element (PPRE) in the caveolin-1 promoter, which was activated upon rosiglitazone treatment in THP-1 macrophages. Conclusions. PPAR activators, such as the PPAR* agonist rosiglitazone, increase caveolin-1 expression in macrophages. This effect appears to be mediated by PPAR activation, possibly by the binding of activated PPAR to the PPRE identified in the caveolin-1 promoter (AU)
