RESUMO
Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.
Assuntos
Autofagia/efeitos dos fármacos , Benzamidas/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Técnicas de Silenciamento de Genes , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fagossomos/efeitos dos fármacos , Fagossomos/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , TransfecçãoRESUMO
Although Imatinib has transformed the treatment of chronic myeloid leukemia (CML), it is not curative due to the persistence of resistant cells that can regenerate the disease. We have examined how Bcr-Abl-expressing cells respond to two mechanistically different therapeutic agents, etoposide and Imatinib. We also examined Bcr-Abl expression at low and high levels as elevated expression has been associated with treatment failure. Cells expressing low levels of Bcr-Abl undergo apoptosis in response to the DNA-targeting agent (etoposide), whereas high-Bcr-Abl-expressing cells primarily induce autophagy. Autophagic populations engage a delayed nonapoptotic death; however, sufficient cells evade this and repopulate following the withdrawal of the drug. Non-Bcr-Abl-expressing 32D or Ba/F3 cells induce both apoptosis and autophagy in response to etoposide and can recover. Imatinib treatment induces both apoptosis and autophagy in all Bcr-Abl-expressing cells and populations rapidly recover. Inhibition of autophagy with ATG7 and Beclin1 siRNA significantly reduced the recovery of Imatinib-treated K562 cells, indicating the importance of autophagy for the recovery of treated cells. Combination regimes incorporating agents that disrupt Imatinib-induced autophagy would remain primarily targeted and may improve response to the treatment in CML.
Assuntos
Autofagia/efeitos dos fármacos , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Proteínas de Fusão bcr-abl/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Autofagia/genética , Benzamidas , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Fusão bcr-abl/genética , Técnicas de Silenciamento de Genes , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Terapia de Alvo Molecular/métodos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Bile salts (BS) have been shown to suppress the secretion of very-low-density lipoprotein-triglyceride (VLDL-TG) in rat and human hepatocytes in vitro. In the present study, we investigated whether the transhepatic BS flux affects VLDL-TG concentration and hepatic VLDL-TG secretion in vivo. In rats, the transhepatic BS flux was quantitatively manipulated by 1-week chronic bile diversion (BD), followed by intraduodenal infusion with taurocholate (TC) or saline for 6 h. In mice, the transhepatic BS flux was manipulated by a 3-week dietary supplementation with TC (0.5 wt.%) or cholestyramine (2 wt.%). In rats, BD followed by saline or TC infusion did not affect plasma triacylglycerol (TG) concentration, hepatic TG production rate or VLDL lipid composition, compared to control rats. In mice supplemented for 3 weeks with TC or cholestyramine, the transhepatic BS flux was increased by 335% and decreased by 48%, respectively, compared to controls. Among the three experimental groups of mice, an inverse relationship between transhepatic BS flux and either plasma TG concentration (R(2)=0.89) or VLDL-TG production rate (R(2)=0.87) was observed, but differences were relatively small. Present data support the concept that BS can reduce VLDL-TG concentration and inhibit hepatic TG secretion in vivo; however, this occurs only at supraphysiological transhepatic BS fluxes in mice.
Assuntos
Ácidos e Sais Biliares/fisiologia , Ductos Biliares Intra-Hepáticos/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Animais , Apolipoproteínas B/biossíntese , Apolipoproteínas B/sangue , Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/metabolismo , Resina de Colestiramina/administração & dosagem , Dieta , Lipoproteínas VLDL/biossíntese , Lipoproteínas VLDL/química , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Ácido Taurocólico/administração & dosagem , Triglicerídeos/análise , Triglicerídeos/biossíntese , Triglicerídeos/sangueRESUMO
Bile salts (BS) inhibit the secretion of apolipoprotein B (apoB) and triacylglycerol (TG) in primary rat, mouse and human hepatocytes and in mice in vivo. We investigated whether lipidation of apoB into a lipoprotein particle is required for this inhibitory action of BS. The sodium/taurocholate co-transporting polypeptide (Ntcp) was co-expressed in McArdle-RH7777 (McA-RH7777) cells stably expressing the full-length human apoB100 (h-apoB100, secreted as TG-rich lipoprotein particles) or carboxyl-truncated human apoB18 (h-apoB18, secreted in lipid-free form). The doubly transfected cell lines (h-apoB/r-Ntcp) effectively accumulated taurocholic acid (TC). TC incubation decreased the secretion of endogenous rat apoB100 (-50%) and h-apoB18 (-35%), but did not affect secretion of rat apoA-I. Pulse-chase experiments (35S-methionine) indicated that the impaired secretion of radiolabeled h-apoB18 and h-apoB100 was associated with accelerated intracellular degradation. The calpain protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) partially inhibited intracellular apoB degradation but did not affect the amount of either h-apoB18 or h-apoB100 secreted into the medium, indicating that inhibition of apoB secretion by TC is not due to calpain-dependent proteasomal degradation. We conclude that TC does not inhibit apoB secretion by interference with its lipidation, but rather involves a mechanism dependent on the N-terminal end of apoB.
Assuntos
Apolipoproteínas B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Ácido Taurocólico/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/biossíntese , Apolipoproteínas B/química , Apolipoproteínas B/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Hepatócitos/química , Hepatócitos/metabolismo , Leupeptinas/farmacologia , Lipoproteínas VLDL/metabolismo , Neoplasias Hepáticas Experimentais , Transportadores de Ânions Orgânicos Dependentes de Sódio , Ratos , Simportadores , Ácido Taurocólico/farmacologia , Fatores de Tempo , Transfecção , Triglicerídeos/metabolismoRESUMO
Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Presenilina-1/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Presenilina-1/genética , Inibidores de Proteases/farmacologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptores Tipo I de Interleucina-1/genética , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
BACKGROUND/AIMS: Expression of hepatic bile salt transporters is partly regulated by bile salts via activation of nuclear farnesoid X-activated receptor (Fxr). We investigated the physiological relevance of this regulation by evaluating transporter expression in mice experiencing different transhepatic bile salt fluxes. METHODS: Bile salt flux was manipulated by dietary supplementation with taurocholate (0.5% w/w) or cholestyramine (2% w/w) or by disruption of the cholesterol 7alpha-hydroxylase-gene (Cyp7A(-/-) mice) leading to reduced bile salt pool size. Expression of hepatic transporters was assessed (polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry). RESULTS: Biliary bile salt secretion was increased (+350%) or decreased (-50%) after taurocholate or cholestyramine feeding, respectively, but plasma bile salt concentrations and hepatic Fxr expression were not affected. The bile salt uptake system Na(+)-taurocholate co-transporting polypeptide (Ntcp) and organic anion transporting polypeptide-1 (Oatp1) were down-regulated by taurocholate and not affected by cholestyramine feeding. Cyp7A(-/-) mice did not show altered Ntcp or Oatp1 expression. Canalicular bile salt export pump (Bsep) was up-regulated by 65% in taurocholate-fed mice, and slightly down-regulated in Cyp7A(-/-) mice. CONCLUSIONS: Large variations in hepatic bile salt flux have minor effects on expression of murine Ntcp and Bsep in vivo, suggesting that these transporters are abundantly expressed and able to accommodate a wide range of 'physiological' bile salt fluxes.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/genética , Colesterol 7-alfa-Hidroxilase/genética , Fígado/metabolismo , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Transporte/metabolismo , Colestanotriol 26-Mono-Oxigenase , Proteínas de Ligação a DNA/genética , Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Citoplasmáticos e Nucleares/genética , Esteroide Hidroxilases/genética , Simportadores , Fatores de Transcrição/genéticaRESUMO
BACKGROUND/AIMS: Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and secrete reduced levels of VLDL-TG. METHODS AND RESULTS: We examined the effects of apoE-deficiency on intracellular lipid homeostasis and secretion of triglycerides (TG). We show that intracellular TG turnover and activities of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) are similar in Apoe(-/-) and wild type mice. In addition, apoB synthesis was not decreased in Apoe(-/-) cells. Thus, the accumulation of lipid in these cells is not attributable to perturbed TG turnover, apoB synthesis, and the activities of DGAT and MTP. Inhibition of MTP had a more profound impact on the secretion of VLDL-TG from wild type hepatocytes than Apoe(-/-) hepatocytes, indicating that MTP was more limiting for the production of VLDL-TG from wild type cells. In marked contrast to the MTP-deficient model of fatty liver, electron microscopy of lipid-stained liver sections of Apoe(-/-) mice revealed an accumulation of lipid in numerous small, putative ER-derived vesicles and in the cytosol. No abnormalities were observed in the Golgi of Apoe(-/-) mice. CONCLUSIONS: These results suggest that the removal of lipids from the early or intermediary compartments of the secretory pathway of hepatocytes is impaired in Apoe(-/-) mice.
Assuntos
Apolipoproteínas E/deficiência , Hepatócitos/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteínas E/genética , Transporte Biológico Ativo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Hepatócitos/ultraestrutura , Lipoproteínas VLDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia EletrônicaRESUMO
The oxysterol-activated liver X receptor (LXR) provides a link between sterol and fatty acid metabolism; activation of LXR induces transcription of lipogenic genes. This study shows that induction of the lipogenic genes Srebp-1c, Fas, and Acc1 upon administration of the synthetic LXR agonist T0901317 to C57BL/6J mice (10 mg/kg/day, 4 days) is associated with massive hepatic steatosis along the entire liver lobule and a 2.5-fold increase in very low density lipoprotein-triglyceride (VLDL-TG) secretion. The increased VLDL-TG secretion was fully accounted for by formation of larger (129 +/- 9 nm versus 94 +/- 12 nm, a 2.5-fold increase of particle volume) TG-rich particles. Stimulation of VLDL-TG secretion did not lead to elevated plasma TG levels in C57BL/6J mice, indicating efficient particle metabolism and clearance. However, T0901317 treatment did lead to severe hypertriglyceridemia in mouse models of defective TG-rich lipoprotein clearance, i.e. APOE*3-Leiden transgenic mice (3.2-fold increase) and apoE-/- LDLr-/- double knockouts (12-fold increase). Incubation of rat hepatoma McA-RH7777 cells with T0901317 also resulted in intracellular TG accumulation and enhanced TG secretion. We conclude that, in addition to raising high density lipoprotein cholesterol concentrations, pharmacological LXR activation in mice leads to development of hepatic steatosis and secretion of atherogenic, large TG-rich VLDL particles.