RESUMO
Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.
Assuntos
Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Aumento de Peso , Tecido Adiposo/metabolismo , Animais , Homeostase , Receptores X do Fígado , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Receptores Nucleares Órfãos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteínas de Ligação a RNA , Receptores X de Retinoides/metabolismoRESUMO
Hyperhomocysteinemia (hHcys) enhances ceramide production, leading to the activation of NADPH oxidase and consequent glomerular oxidative stress and sclerosis. The present study was performed to determine whether acid sphingomyelinase (Asm), a ceramide-producing enzyme, is implicated in the development of hHcys-induced glomerular oxidative stress and injury. Uninephrectomized Asm-knockout (Asm(-/-)) and wild-type (Asm(+/+)) mice, with or without Asm short hairpin RNA (shRNA) transfection, were fed a folate-free (FF) diet for 8 weeks, which significantly elevated the plasma Hcys level compared with mice fed normal chow. By using in vivo molecular imaging, we found that transfected shRNAs were expressed in the renal cortex starting on day 3 and continued for 24 days. The FF diet significantly increased renal ceramide production, Asm mRNA and activity, urinary total protein and albumin excretion, glomerular damage index, and NADPH-dependent superoxide production in the renal cortex from Asm(+/+) mice compared with that from Asm(-/-) or Asm shRNA-transfected wild-type mice. Immunofluorescence analysis showed that the FF diet decreased the expression of podocin but increased desmin and ceramide levels in glomeruli from Asm(+/+) mice but not in those from Asm(-/-) and Asm shRNA-transfected wild-type mice. In conclusion, our observations reveal that Asm plays a pivotal role in mediating podocyte injury and glomerular sclerosis associated with NADPH oxidase-associated local oxidative stress during hHcys.
Assuntos
Inativação Gênica/fisiologia , Hiper-Homocisteinemia/complicações , Nefropatias/enzimologia , Glomérulos Renais/enzimologia , Estresse Oxidativo/genética , Esfingomielina Fosfodiesterase/genética , Animais , Células Cultivadas , Ceramidas/metabolismo , Técnicas de Transferência de Genes , Nefropatias/genética , Camundongos , Camundongos Endogâmicos C57BL , Microbolhas , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/metabolismo , Superóxidos/metabolismoRESUMO
The pathogenesis of hepatic encephalopathy(HE) is unclear. However gut flora changes, inflammation and neuro-glial injury have been implicated. The aim was to evaluate factors that were associated with HE recurrence after lactulose withdrawal by analyzing the clinical phenotype, stool microbiome and systemic metabolome longitudinally. HE patients on a standard diet who were adherent on lactulose underwent characterization of their phenotype [cognition, inflammatory cytokines, in-vivo brain MR spectroscopy(MRS)], gut microbiome (stool Multitag Pyrosequencing) and metabolome (urine/serum ex-vivo MRS) analysis while on lactulose and on days 2, 14 and 30 post-withdrawal. Patients whose HE recurred post-withdrawal were compared to those without recurrence. We included seven men (53 ± 8 years) who were adherent on lactulose after a precipitated HE episode were included. HE recurred in three men 32 ± 6 days post-withdrawal. In-vivo brain MRS showed increased glutamine+glutamate (Glx) and decreased myoinositol with a reduction in stool Faecalibacterium spp., post-withdrawal. HE recurrence was predicted by poor baseline inhibitory control and block design performance and was associated with a shift of choline metabolism from tri-methylamine oxide formation towards the development of di-methylglycine, glycine and creatinine. This was accompanied by a mixed effect on the immune response (suppressed IL-10 and Th1/Th2/Th17 response). The correlation network showed Prevotella to be linked to improved cognition and decreased inflammation in patients without HE recurrence. We conclude that lactulose withdrawal results in worsening cognition, mixed inflammatory response effect, lowered stool Faecalibacterium and increase in MR-measurable brain Glx. HE recurrence post-lactulose withdrawal can be predicted by baseline cognitive performance and is accompanied by disrupted choline metabolism.
Assuntos
Encefalopatia Hepática/fisiopatologia , Lactulose/farmacologia , Biologia de Sistemas/métodos , Idoso , Química Encefálica , Citocinas/sangue , Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Encefalopatia Hepática/tratamento farmacológico , Encefalopatia Hepática/psicologia , Humanos , Análise dos Mínimos Quadrados , Estudos Longitudinais , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Metagenoma , Pessoa de Meia-Idade , Testes Neuropsicológicos , RecidivaRESUMO
Ansamycin antibiotics that target heat shock protein 90 function are being developed as anticancer agents but are also known to be dose limiting in patients due to hepatotoxicity. Herein, to better understand how the normal tissue toxicity of geldanamycins could be ameliorated to improve the therapeutic index of these agents, we examined the interactions of 17-allylamino-17-demethoxygeldanamycin (17AAG) and the secondary bile acid deoxycholic acid (DCA) in hepatocytes and fibroblasts. DCA and 17AAG interacted in a greater than additive fashion to cause hepatocyte cell death within 2 to 6 h of coadministration. As single agents DCA, but not 17AAG, enhanced the activity of extracellular signal-regulated kinase 1/2, AKT, c-Jun NH(2)-terminal kinase 1/2 (JNK1/2), and p38 mitogen-activated protein kinase (MAPK). Combined exposure of cells to DCA and 17AAG further enhanced JNK1/2 and p38 MAPK activity. Inhibition of JNK1/2 or p38 MAPK, but not activator protein-1, suppressed the lethality of 17AAG and of 17AAG and DCA. Constitutive activation of AKT, but not MAPK/extracellular signal-regulated kinase kinase 1/2, suppressed 17AAG- and DCA-induced cell killing and reduced activation of JNK1/2. DCA and 17AAG exposure promoted association of BAX with mitochondria, and functional inhibition of BAX or caspase-9, but not of BID and caspase-8, suppressed 17AAG and DCA lethality. DCA and 17AAG interacted in a greater than additive fashion to promote and prolong the generation of reactive oxygen species (ROS). ROS-quenching agents, inhibition of mitochondrial function, expression of dominant-negative thioredoxin reductase, or expression of dominant-negative apoptosis signaling kinase 1 suppressed JNK1/2 and p38 MAPK activation and reduced cell killing after 17AAG and DCA exposure. The potentiation of DCA-induced ROS production by 17AAG was abolished by Ca(2+) chelation and ROS generation, and cell killing following 17AAG and DCA treatment was abolished in cells lacking expression of PKR-like endoplasmic reticulum kinase. Thus, DCA and 17AAG interact to stimulate Ca(2+)-dependent and PKR-like endoplasmic reticulum kinase-dependent ROS production; high levels of ROS promote intense activation of the p38 MAPK and JNK1/2 pathways that signal to activate the intrinsic apoptosis pathway.
Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Ácido Desoxicólico/farmacologia , Detergentes/farmacologia , Hepatócitos/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Cálcio , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Ceramidas/metabolismo , Quimioterapia Combinada , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun NH(2)-terminal kinase (JNK) pathways, and downstream transcription factors, in the survival response of hepatocytes. DCA caused activation of the ERK1/2 and JNK1/2 pathways. Inhibition of either DCA-induced ERK1/2 or DCA-induced JNK1/2 signaling enhanced the apoptotic response of hepatocytes. Further analyses demonstrated that DCA-induced JNK2 signaling was cytoprotective whereas DCA-induced JNK1 signaling was cytotoxic. DCA-induced ERK1/2 activation was responsible for increased DNA binding of C/EBPbeta, CREB, and c-Jun/AP-1. Inhibition of C/EBPbeta, CREB, and c-Jun function promoted apoptosis following DCA treatment, and the level of apoptosis was further increased in the case of CREB and c-Jun, but not C/EBPbeta, by inhibition of MEK1/2. The combined loss of CREB and c-Jun function or of C/EBPbeta and c-Jun function enhanced DCA-induced apoptosis above the levels resulting from the loss of either factor individually; however, these effects were less than additive. Loss of c-Jun or CREB function correlated with increased expression of FAS death receptor and PUMA and decreased expression of c-FLIP-(L) and c-FLIP-(S), proteins previously implicated in the modulation of the cellular apoptotic response. Collectively, these data demonstrate that multiple DCA-induced signaling pathways and transcription factors control hepatocyte survival.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ácido Desoxicólico/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Receptor fas/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
This chapter will describe methods to assess the activities of protein kinases. Initial studies in the 1950s and 1960s in the field of glucose metabolism examined the activities of several highly specific protein and carbohydrate kinases in cell lysates or isolated cell fractions. As more protein kinases were discovered in the 1980s and 1990s, coupled with the development of immunoprecipitating antibodies, in vitro kinase assays of isolated kinase proteins using gamma-32P ATP became a standard procedure. In the 1990s, antibodies were developed that recognize specific sites of regulatory phosphorylation on a variety of protein kinases (phospho-specific antibodies), which have been used to assess kinase activity indirectly through immunoblotting. In this chapter, Methodologies to perform immune complex protein kinase assays and immunoblotting using phospho-specific antibodies against regulatory sites of phosphorylation in protein kinases will be described. The strengths and weaknesses of each approach in determining protein kinase activity will also be discussed.
Assuntos
Neoplasias/metabolismo , Transdução de Sinais , Animais , Anticorpos Fosfo-Específicos , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo , Eletroforese em Gel de Poliacrilamida , Hepatócitos , Masculino , Fosforilação , Testes de Precipitina , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Modulation of ERBB and insulin-like growth factor 1 (IGF-1) receptor function is recognized as a potential mechanism to inhibit tumor growth. We and others have shown that inhibition of ERBB1 can enhance bile acid toxicity. Herein, we extend our analyses to examine the impact of insulin/IGF-1 receptor inhibition on primary hepatocyte survival when exposed to the secondary bile acid deoxycholic acid (DCA) and compare the impact inhibition of this receptor has on bile acid toxicity effects to that of ERBB1/MEK1/2 inhibition. The insulin/IGF-1 receptor inhibitor NVP-ADW742 at concentrations which inhibit both the insulin and IGF-1 receptors had a modest negative impact on hepatocyte viability, and strongly potentiated DCA-induced apoptotic cell death. Identical data were obtained expressing a dominant negative IGF-1 receptor in hepatocytes; a receptor which acts to inhibit both the IGF-1 receptor and the insulin receptor in trans. Inhibition of ERBB1 function using Iressa (gefitinib) or the tyrphostin AG1478 had more modest effects at enhancing DCA lethality than inhibition of the insulin/IGF-1 receptor function. In contrast, over-expression of a dominant negative ERBB1 protein had pleiotropic effects on multiple signaling pathways in an apparently non-specific manner. These findings suggest that novel therapeutic kinase inhibitors, targeted against growth factor receptors, have the potential to promote bile acid toxicity in hepatocyte when bile flow may be impaired.
Assuntos
Ácidos e Sais Biliares/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Antagonistas da Insulina/farmacologia , Insulina/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ácido Desoxicólico/toxicidade , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/metabolismoRESUMO
The manuscripts by Park et al. and Zhang et al. were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor-induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2alpha-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Ceramidas/metabolismo , Neoplasias/tratamento farmacológico , eIF-2 Quinase/metabolismo , Receptor fas/metabolismo , Apoptose , Caspase 8/metabolismo , Humanos , Transdução de SinaisRESUMO
Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Colagogos e Coleréticos/farmacologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Ácido Desoxicólico/farmacologia , Hepatócitos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia , Bile/metabolismo , Células Cultivadas , Proteínas Inibidoras de Quinase Dependente de Ciclina/antagonistas & inibidores , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Ácido Desoxicólico/metabolismo , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica , Hepatócitos/citologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Receptor fas/genéticaRESUMO
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2alpha phosphorylation; suppression of eIF2alpha function abolished drug combination lethality. Cell killing was paralleled by PERK-and eIF2alpha-dependent lowering of c-FLIP-s protein levels and overexpression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRbeta and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Overexpression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2alpha- downward arrowc-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
Assuntos
Antineoplásicos/farmacologia , Autofagia , Benzenossulfonatos/farmacologia , Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Hidroxâmicos/farmacologia , Piridinas/farmacologia , eIF-2 Quinase/biossíntese , Receptor fas/biossíntese , Caspase 8/metabolismo , Sobrevivência Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Modelos Biológicos , Mutação , Niacinamida/análogos & derivados , Compostos de Fenilureia , Sorafenibe , Resultado do Tratamento , VorinostatRESUMO
The regulation of glycogen synthase activity by bile acids in primary hepatocytes and in the intact liver was investigated. Bile acids (deoxycholic acid, DCA; taurocholic acid, TCA) activated AKT and glycogen synthase (GS) in primary rat hepatocytes. Incubation with a phosphatidyl inositol-3 kinase inhibitor or expression of dominant-negative AKT in primary rat hepatocytes abolished activation of AKT and GS by DCA and TCA. TCA, but not DCA, activated Galpha(i) proteins in primary rat hepatocytes. Treatment of cells with pertussis toxin or expression of dominant-negative Galpha(i) blocked TCA-induced activation of AKT and of GS but did not alter AKT or GS activation caused by DCA. TCA caused activation of AKT and GS in intact rat liver. Expression of dominant-negative Galpha(i) reduced TCA-induced activation of AKT and of GS in intact rat liver. Together, our findings demonstrate that bile acids are physiological regulators of glycogen synthase in rat liver and that conjugated bile acids use a Galpha(i)-coupled G protein-coupled receptor to regulate GS activity in vitro and in vivo.
Assuntos
Ácidos e Sais Biliares/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Glicogênio Sintase/metabolismo , Hepatócitos/enzimologia , Transdução de Sinais , Animais , Células Cultivadas , Ácido Desoxicólico/farmacologia , Hepatócitos/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido Taurodesoxicólico/farmacologiaRESUMO
Several studies have argued that G-protein-coupled receptors (GPCR) have the capacity to promote activation of receptor tyrosine kinases. The current studies were performed to examine the regulation of the extracellular regulated kinase (ERK)1/2 and AKT pathways by conjugated and unconjugated bile acids in primary hepatocytes. Deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), glycocholic acid (GCA), and tauroursodeoxycholic acid (TUDCA) all activated ERK1/2 in primary rat hepatocytes that was abolished by inhibition of ERBB1, and significantly reduced by ROS quenching agents. Bile acid-induced AKT activation was blunted by preventing ERBB1 activation and ROS generation. Treatment of rat hepatocytes with pertussis toxin (PTX) did not alter ERK1/2 and AKT activation induced by DCA or CDCA but abolished pathway activations by conjugated bile acids. Similar data to those with PTX were obtained when a dominant negative form of G(i1alpha) was overexpressed. Treatment of rat hepatocytes with TDCA and TCA promoted guanosine triphosphate (GTP) loading of G(i1alpha), G(i2alpha), and G(i3alpha) in vitro. Treatment of rat hepatocytes with PTX abolished TDCA-induced tyrosine phosphorylation of ERBB1. Similar findings to those in rat hepatocytes were also obtained in primary mouse and human hepatocytes, but not in established rodent or human hepatoma cell lines. In conclusion, collectively our findings demonstrate that unconjugated bile acids activate hepatocyte receptor tyrosine kinases and intracellular signaling pathways in a ROS-dependent manner. In contrast, conjugated bile acids primarily activate receptor tyrosine kinases and intracellular signaling pathways in a GPCR (G(ialpha))-dependent and ROS-dependent manner.
Assuntos
Ácidos e Sais Biliares/farmacologia , Hepatócitos/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Toxina Pertussis/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ácido Glicoquenodesoxicólico/farmacologia , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , Ácido Taurodesoxicólico/farmacologiaRESUMO
In the current study, we investigated the effect of growth of FasL(+) tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL(+) LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vbeta3(+) and Vbeta11(+) T cells. When injected into syngeneic mice, the FasL(+) LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin beta, and tumor necrosis factor beta. Together, these data suggested that, in mice bearing FasL(+) LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.
Assuntos
Apoptose/imunologia , Fígado/imunologia , Linfoma de Células T/imunologia , Glicoproteínas de Membrana/imunologia , Baço/imunologia , Animais , Aspartato Aminotransferases/sangue , Divisão Celular , Citocinas/genética , Citocinas/imunologia , Proteína Ligante Fas , Feminino , Citometria de Fluxo , Hepatócitos/imunologia , Marcação In Situ das Extremidades Cortadas , Fígado/patologia , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos MRL lpr , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Baço/patologia , Superantígenos/imunologia , Linfócitos T/imunologiaRESUMO
The mechanisms by which bile acids induce apoptosis in hepatocytes and the signaling pathways involved in the control of cell death are not understood fully. Here, we examined the impact of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K) signaling on the survival of primary hepatocytes exposed to bile acids. Treatment of hepatocytes with deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA) caused sustained MAPK activation that was dependent on activation of the epidermal growth factor receptor (EGFR). Activation of MAPK was partially blocked by inhibitors of PI3K. Inhibition of DCA-, CDCA-, and UDCA-stimulated MAPK activation resulted in approximately 20%, approximately 35%, and approximately 55% apoptosis, respectively. The potentiation of DCA- and CDCA-induced apoptosis by MEK1/2 inhibitors correlated with cleavage of procaspase 3, which was blocked by inhibitors of caspase 8 (ile-Glu-Thr-Asp-p-nitroanilide [IETD]) and caspase 3 (DEVD). In contrast, the potentiation of UDCA-induced apoptosis weakly correlated with procaspase 3 cleavage, yet this effect was also blocked by IETD and DEVD. Incubation of hepatocytes with the serine protease inhibitor AEBSF reduced the death response of cells treated with UDCA and MEK1/2 inhibitor to that observed for DCA and MEK1/2 inhibitor. The apoptotic response was FAS receptor- and neutral sphingomyelinase-dependent and independent of FAS ligand expression, and neither chelation of intracellular and extracellular Ca(2+) nor down-regulation of PKC expression altered the apoptotic effects of bile acids. In conclusion, bile acid apoptosis is dependent on the production of ceramide and is counteracted by activation of the MAPK and PI3K pathways.
Assuntos
Apoptose/fisiologia , Colagogos e Coleréticos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Ácido Ursodesoxicólico/farmacologia , Animais , Caspase 3 , Caspases/química , Caspases/efeitos dos fármacos , Células Cultivadas , Ácido Quenodesoxicólico/farmacologia , Ácido Desoxicólico/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/química , Precursores Enzimáticos/efeitos dos fármacos , Receptores ErbB/fisiologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
Previously, we demonstrated that deoxycholic acid (DCA)-induced ERK1/2 and AKT signaling in primary hepatocytes is a protective response. In the present study, we examined the regulation of the phosphatidylinositol 3 (PI3) kinase/AKT/glycogen synthase (kinase) 3 (GSK3)/glycogen synthase (GS) pathway by bile acids. In primary hepatocytes, DCA activated ERBB1 (the epidermal growth factor receptor), ERBB2, and the insulin receptor, but not the insulin-like growth factor 1 (IGF-1) receptor. DCA-induced activation of the insulin receptor correlated with enhanced phosphorylation of insulin receptor substrate 1, effects that were both blocked by the insulin receptor inhibitor AG1024 and by expression of the dominant negative IGF-1 receptor (K1003R), which inhibited in trans. Expression of the dominant negative IGF-1 receptor (K1003R) also abolished DCA-induced AKT activation. Bile acid-induced activation of AKT and phosphorylation of GSK3 were blunted by the ERBB1 inhibitor AG1478 and abolished by AG1024. Bile acids caused activation of GS to a similar level induced by insulin (50 nM); both were blocked by inhibition of insulin receptor function and the PI3 kinase/AKT/GSK3 pathway. In conclusion, these findings suggest that bile acids and insulin may cooperate to regulate glucose storage in hepatocytes.
Assuntos
Ácidos e Sais Biliares/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase/metabolismo , Hepatócitos/enzimologia , Proteínas Serina-Treonina Quinases , Receptor IGF Tipo 1/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes erbB-1/fisiologia , Genes erbB-2/fisiologia , Vetores Genéticos , Glucose/metabolismo , Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/genéticaRESUMO
Prolonged activation of the mitogen-activated protein kinase (MAPK) pathway enhances expression of the cyclin kinase inhibitor p21 that can promote growth arrest and cell survival in response to cytotoxic insults. Bile acids can also cause prolonged MAPK activation that is cytoprotective against bile acid-induced cell death. Here, we examined the impact of bile acid-induced MAPK signaling and p21 expression on the survival of primary mouse hepatocytes. Deoxycholic acid (DCA) caused prolonged activation of the MAPK pathway that weakly enhanced p21 protein expression. When DCA-induced MAPK activation was blocked using MEK1/2 inhibitors, both hepatocyte viability and expression of p21 were reduced. Surprisingly, constitutive overexpression of p21 in p21+/+ hepatocytes enhanced DCA-induced cell killing. In agreement with these findings, treatment of p21-/- hepatocytes with DCA and MEK1/2 inhibitors also caused less apoptosis than observed in wild-type p21+/+ cells. Expression of p21 in p21-/- hepatocytes did not modify basal levels of apoptosis but restored the apoptotic response of p21-/- cells to those of p21+/+ cells overexpressing p21. These findings suggest that basal expression of p21 plays a facilitating, proapoptotic role in DCA-induced apoptosis. Overexpression of p21 enhanced p53 protein levels. In agreement with a role for p53 in the enhanced apoptotic response, overexpression of p21 did not potentiate apoptosis in p53-/- hepatocytes but, instead, attenuated the death response in these cells. In conclusion, our data suggest that overexpression of p21 can promote apoptosis, leading to elevated sensitivity to proapoptotic stimuli.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Ciclinas/fisiologia , Hepatócitos/citologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Western Blotting , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/deficiência , Ciclinas/genética , Ácido Desoxicólico/farmacologia , Sinergismo Farmacológico , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Receptores ErbB/metabolismo , Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
BACKGROUND: The hepatitis B virus X gene has three in-frame start codons encoding the pX, AUG2 and AUG3 proteins. The AUG2 and AUG3 genes are 5'-truncated in respect to the full-length pX gene; however, all three genes terminate at the same stop codon. The activity of pX as an oncogene is well characterized; however, less is known about the AUG2 and AUG3 proteins. METHODS: The effects of pX, AUG2 and AUG3 on p21Cip,1/WAF,1/MDA6 and p27Kip-1 cyclin kinase inhibitor (CKI) protein expression, and the impact they have on proliferation, were investigated in CHO K-1 cells. CHO K-1 cells were chosen because they can be transfected at 100% efficiency. RESULTS: p21- and p27-luciferase reporter expression is modulated by increasing doses of the hepatitis B X proteins. At low concentrations of pX or AUG2, p21- and p27-luciferase activity was increased, and at high concentrations, p21- and p27-luciferase activity was decreased. Expression of the AUG3 gene showed a different profile: it was increasingly stimulatory with dose for both promoters. Western blot analyses demonstrated that p21 and p27 protein levels were modulated as predicted based on data generated in the promoter-luciferase experiments. Tritiated thymidine labeling of DNA showed biphasic kinetics of incorporation in the presence of varying pX and AUG2 concentrations, whereas labeling decreased with AUG3 concentration. The growth inhibitory effect of pX expression was reduced by antisense ablation of either p21 or p27. CONCLUSIONS: The relative expression level of pX, AUG2, and AUG3 impacts on CKI expression and cell proliferation. Our findings may explain why divergent effects of pX expression on growth have been observed by different groups, which may be related to relative pX expression levels.