RESUMO
Nucling is a stress-inducible protein associated with apoptosomes. The cytochrome c-triggered formation of apoptosomes represents a key-initiating event in apoptosis. We have recently reported that Nucling regulates the apoptotic pathway by controlling the activation of NF-kappaB as well. Here we show that hepatocellular carcinoma (HCC) arising spontaneously against a background of hepatitis occurred more frequently in Nucling-knockout (KO) mice than wild-type (WT) mice. Biochemical serum testing revealed potential liver dysfunction with hypercholesterolemia in Nucling-KO males. In the background of Nucling-KO mice, we observed the up-regulation of TNFalpha, spontaneous NF-kappaB-activation and the induction of galectin-3 expression in liver. In addition, we observed a decrease in the number of Kupffer cells (KCs) in the KO mice. KCs are important for the hepatic immune system, acting as phagocytes or antigen-presenting cells (APCs). We found that KCs in Nucling-KO mice were apoptotic possibly through the up-regulation of TNFalpha. These observations indicate that Nucling is important for the regulation of NF-kappaB signals in liver. We propose that Nucling deficiency could be a powerful tool to reveal the NF-kappaB-related molecular networks leading to hepatitis and HCC development.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Hepatite/genética , Células de Kupffer/patologia , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Animais , Western Blotting , Tetracloreto de Carbono/toxicidade , Carcinógenos/toxicidade , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Hepatite/complicações , Hepatite/patologia , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nucling is an Apaf1-binding proapoptotic protein involved in apoptosome-mediated apoptosis. Luciferase assays have revealed that the activation of nuclear factor-kappaB induced by tumor necrosis factor-alpha, interleukin-1beta and lipopolysaccharide is downregulated by the overexpression of Nucling in HEK293 cells. Moreover, the expression of endogenous cyclooxygenase 2, tumor necrosis factor-alpha and galectin-3, the end-point molecules in the pathway for the activation of nuclear factor-kappaB, as well as nuclear factor-kappaB (p65) itself, is upregulated in Nucling gene-deficient mouse embryonic fibroblasts, suggesting that nuclear factor-kappaB is a target of Nucling. Subsequent study has revealed that Nucling physically interacts with nuclear factor-kappaB (p65 and p50) and that the binding domain of Nucling is its amino-terminal region (amino acids 1-466) containing ankyrin repeats. Overexpression of Nucling prevents the translocation of nuclear factor-kappaB into the nucleus. In addition, the cytoplasmic retention of endogenous nuclear factor-kappaB in resting cells is not observed in Nucling gene-deficient mouse embryonic fibroblasts. These results reveal a novel function of Nucling as a suppressor of nuclear factor-kappaB, mediated by its cytoplasmic retention through physical interaction.
Assuntos
Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Animais , Células COS , Núcleo Celular , Células Cultivadas , Chlorocebus aethiops , Citoplasma , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.