RESUMEN
Nuclear factor of activated T cells 5 protein (NFAT5) is thought to be important for cellular adaptation to osmotic stress by regulating the transcription of genes responsible for the synthesis or transport of organic osmolytes. It is also thought to play a role in immune function, myogenesis and cancer invasion. To better understand the function of NFAT5, we developed NFAT5 gene knockout mice. Homozygous NFAT5 null (NFAT5(-/-)) mouse embryos failed to develop normally and died after 14.5 days of embryonic development (E14.5). The embryos showed peripheral edema, and abnormal heart development as indicated by thinner ventricular wall and reduced cell density at the compact and trabecular areas of myocardium. This is associated with reduced level of proliferating cell nuclear antigen and increased caspase-3 in these tissues. Cardiomyocytes from E14.5 NFAT5(-/-) embryos showed a significant reduction of beating rate and abnormal Ca(2+) signaling profile as a consequence of reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and ryanodine receptor (RyR) expressions. Expression of NFAT5 target genes, such as HSP 70 and SMIT were reduced in NFAT5(-/-) cardiomyocytes. Our findings demonstrated an essential role of NFAT5 in cardiac development and Ca(2+) signaling. Cardiac failure is most likely responsible for the peripheral edema and death of NFAT5(-/-) embryos at E14.5 days.
Asunto(s)
Pérdida del Embrión/patología , Pérdida del Embrión/fisiopatología , Corazón/embriología , Corazón/fisiopatología , Factores de Transcripción/deficiencia , Animales , Apoptosis , Señalización del Calcio , Anomalías Cardiovasculares/complicaciones , Anomalías Cardiovasculares/patología , Anomalías Cardiovasculares/fisiopatología , Proliferación Celular , Regulación hacia Abajo/genética , Edema/complicaciones , Edema/patología , Pérdida del Embrión/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Marcación de Gen , Vectores Genéticos/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Pruebas de Función Cardíaca , Espacio Intracelular/metabolismo , Ratones , Ratones Mutantes , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Simportadores/genética , Simportadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.