RESUMO
Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.
Assuntos
Endorribonucleases , Grânulos de Estresse , Proteínas Ativadoras de ras GTPase , Arsenitos/farmacologia , Endorribonucleases/genética , Proteínas Ativadoras de GTPase/genética , RNA Mensageiro/genéticaRESUMO
We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation.
Assuntos
Fase G1 , Hepatócitos/citologia , Quinase de Cadeia Leve de Miosina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fase S , Amidas/farmacologia , Animais , Azepinas/farmacologia , Northern Blotting , Células Cultivadas , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptor Quinase 1 Acoplada a Proteína G/efeitos dos fármacos , Hepatócitos/metabolismo , Técnicas In Vitro , Masculino , Microscopia de Fluorescência , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Naftalenos/farmacologia , Piridinas/farmacologia , RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/genéticaRESUMO
BACKGROUND/AIMS: Patients exhibiting hepatic iron overload frequently develop hepatocellular carcinoma. An impaired expression of hepatic genes could be involved in this phenomenon. Our aim was to identify, during iron overload, hepatic genes involved in cell cycle which are misregulated. RESULTS: Mouse iron overload was obtained by carbonyl-iron supplementation or iron-dextran injection. As expected, liver iron overload was associated to both hepatomegaly and hepatocyte polyploidisation. Hepatic gene expression was investigated using macroarray hybridizations. Cyclin D1 mRNA was the only gene whose expression increased in both models. Its overexpression was confirmed by real-time quantitative PCR. Immunobloting analysis demonstrated a strong increase of Cyclin D1 protein expression in iron-overloaded hepatocytes. This overexpression was correlated with early abnormalities in their cell cycle progression judged, in vitro, on DNA synthesis and mitotic index increase. CONCLUSIONS: Our data demonstrates that Cyclin D1, a protein involved in G1-phase of cell cycle, is overexpressed in the iron-overloaded liver. This iron-induced expression of Cyclin D1 may contribute to development of cell cycle abnormalities, suggesting a role of Cyclin D1 in iron-related hepatocarcinogenesis.
Assuntos
Ciclo Celular/genética , Ciclina D1/genética , Expressão Gênica , Hepatócitos/patologia , Sobrecarga de Ferro , RNA Mensageiro/genética , Animais , Western Blotting , Células Cultivadas , Ciclina D1/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Hepatócitos/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepcidin, which has been recently identified both by biochemical and genomic approaches, is a 25 amino acid polypeptide synthesized mainly by hepatocytes and secreted into the plasma. Besides its potential activity in antimicrobial defense, hepcidin plays a major role in iron metabolism. It controls two key steps of iron bioavailability, likely through a hormonal action: digestive iron absorption by enterocytes and iron recycling by macrophages. In humans, this could explain that low levels of hepcidin found during juvenile haemochromatosis and HFE-1 genetic haemochromatosis are associated with an iron overload phenotype. Conversely, an increase of hepcidin expression is suspected to play a major role in the development of anemia of chronic inflammatory diseases. However, the regulatory mechanisms of hepcidin expression are multiple, including iron-related parameters, anemia, hypoxia, inflammation and hepatocyte function. Therefore, many physiological and pathological situations may modulate hepcidin expression and subsequently iron metabolism. A better knowledge of the biological effects of hepcidin and of its expression regulatory mechanisms will clarify the place of hepcidin in the diagnosis and treatment of iron-related diseases.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Ferro/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Expressão Gênica , Hepatócitos/metabolismo , Hepcidinas , Humanos , Infecções/imunologia , Inflamação/imunologia , Hepatopatias/metabolismo , Modelos Biológicos , Dados de Sequência MolecularRESUMO
Hepcidin, a key regulator of iron metabolism, is synthesized by the liver. Hepcidin binds to the iron exporter ferroportin to regulate the release of iron into plasma from macrophages, hepatocytes, and enterocytes. We analyzed liver samples from patients undergoing hepatic surgery for cancer or receiving liver transplants and analyzed correlations between clinical parameters and liver hepcidin mRNA and urinary hepcidin concentrations. Despite the many potential confounding influences, urinary hepcidin concentrations significantly correlated with hepatic hepcidin mRNA concentrations, indicating that hepcidin quantification in urine is a valid approach to evaluate hepcidin expression. Moreover, we found in humans that hepcidin levels correlated with hepatic iron stores and hemoglobin levels and may also be affected by hepatic dysfunction.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/urina , Hemoglobinas/metabolismo , Fígado/fisiologia , Adulto , Idoso , Sequência de Bases , DNA/genética , Feminino , Hepcidinas , Humanos , Cirrose Hepática/genética , Cirrose Hepática/fisiopatologia , Cirrose Hepática/urina , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
During liver regeneration, hepatocytes proliferate under the control of both proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) and growth factors, in parallel to extracellular matrix remodeling. This study investigated mechanisms by which mitogen and extracellular matrix signals are linked for inducing proliferation of differentiated hepatocytes. The authors used adult rat hepatocytes in coculture with liver biliary cells, because cells are stably differentiated for several weeks, capable of extracellular matrix deposition, and unable to divide in response to growth factor alone. This work demonstrated that hepatocytes could undergo several proliferation waves without loss of differentiation by using alternating periods of TNFalpha/growth factor stimulation and deprivation. Three days after stimulation with TNFalpha and epidermal growth factor (EGF), up to 35% of hepatocytes divided. Demonstration was also provided that EGF alone only promoted cell progression up to late G(1), whereas TNFalpha was necessary for G(1)/S transition and Cdk1 induction. TNFalpha promoted an extracellular matrix (ECM) degradation that involved the matrix metalloproteinase MMP-9 induction through activation of NF-kappaB pathway. Finally, the authors showed that ECM remodeling signal was required for initiating any new hepatocyte division wave, in presence of mitogen. In conclusion, these results highlight that hepatocyte division is dependent on ECM deposition associated with differentiation status, and that ECM degradation signal is critical in controlling G(1)/S transition and Cdk1 induction. These results provide new insights for understanding the unique hepatocyte proliferation control and improving regeneration in patients suffering from liver damage.
Assuntos
Matriz Extracelular/metabolismo , Hepatócitos/citologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteína Quinase CDC2/biossíntese , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Masculino , Metaloproteinase 9 da Matriz/genética , NF-kappa B/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Hepcidin (HEPC) plays a key role in iron homeostasis and an abnormally low level of hepcidin mRNA has been reported in HFE-1 genetic hemochromatosis. Considering the well-known phenotypic variability of this disease, especially between men and women, it is important to define factors susceptible to modulate hepatic hepcidin expression and, consequently, to influence the development of iron overload in HFE-1 hemochromatosis. Therefore, our aim was to analyze the effects of strain and gender on hepatic hepcidin expression in the mouse. C57BL/6 and DBA/2 wild-type mice were included in this study. Liver and splenic iron contents were measured. Specific hepatic Hepc1 and Hepc2 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). C57BL/6 mice expressed predominantly Hepc1 mRNA, whereas Hepc2 mRNA was the main form in DBA/2 mice. In both strains, females had higher levels of iron stores and Hepc mRNAs compared to males. Our results demonstrate that the expression of both hepcidin mRNAs varies according to strain and gender. They suggest that sex and genetic background, which are regulators of hepcidin expression, could play a role in the phenotypic expression of genetic hemochromatosis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Regulação da Expressão Gênica , Fígado/química , Animais , Suscetibilidade a Doenças , Hemocromatose/etiologia , Hepcidinas , Ferro/análise , Sobrecarga de Ferro/etiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , RNA Mensageiro/análise , Fatores Sexuais , Baço/químicaRESUMO
In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A-particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl-iron and iron-dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Sobrecarga de Ferro/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Sequência de Bases , Componentes do Gene , Regulação da Expressão Gênica , Hepcidinas , Sobrecarga de Ferro/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Distribuição TecidualRESUMO
Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.
Assuntos
Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/enzimologia , Endorribonucleases/metabolismo , Células Eucarióticas/enzimologia , RNA Mensageiro/metabolismo , Estresse Fisiológico/enzimologia , Proteínas Ativadoras de ras GTPase/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Arseniatos/farmacologia , Células COS , Proteínas de Transporte/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Grânulos Citoplasmáticos/genética , DNA Helicases , Endorribonucleases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HeLa , Humanos , Mutação/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Serina/metabolismo , Estresse Fisiológico/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.