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BACKGROUND & AIMS: Ubiquitination is a reversible protein modification involved in the major cellular processes that define cell phenotype and behaviour. Ubiquitin modifications are removed by a large family of proteases named deubiquitinases. The role of deubiquitinases in hepatic stellate cell (HSC) activation and their contribution to fibrogenesis are poorly defined. We have identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, determined its function in activated HSC and its potential as a therapeutic target for fibrosis. METHODS: Deubiquitinase expression was determined in day 0 and day 10 HSC. Increased UCHL1 expression was confirmed in human HSC and in an alcoholic liver disease (ALD) patient liver. The importance of UCHL1 in hepatic fibrosis was investigated in CCl4 and bile duct ligation injured mice using a pharmacological inhibitor (LDN 57444). The effects of UCHL1 inhibition on HSC proliferation were confirmed by Western blot and 3H thymidine incorporation. RESULTS: Here we report that pharmacological inhibition of UCHL1 blocks progression of established fibrosis in CCl4 injured mice. UCHL1 siRNA knockdown, LDN 57444 treatment, or HSC isolated from UCHL1(-/-) mice show attenuated proliferation in response to the mitogen, platelet-derived growth factor. Additionally, we observed changes in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb) in the absence of UCHL1 highlighting a potential mechanism for the reduced proliferative response. CONCLUSIONS: UCHL1 expression is highly upregulated upon HSC activation and is involved in the regulation of HSC proliferation. This study highlights therapeutic opportunities for pharmacological targeting of UCHL1 in chronic liver disease.
Assuntos
Hepatopatias/enzimologia , Ubiquitina Tiolesterase/metabolismo , Animais , Biomarcadores/metabolismo , Tetracloreto de Carbono/toxicidade , Proliferação de Células , Transdiferenciação Celular , Células Cultivadas , Doença Crônica , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/patologia , Hepatopatias/patologia , Hepatopatias/terapia , Hepatopatias Alcoólicas/enzimologia , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/enzimologia , Miofibroblastos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
UNLABELLED: Toll-like receptors (TLRs) function as key regulators of liver fibrosis and are able to modulate the fibrogenic actions of nonparenchymal liver cells. The fibrogenic signaling events downstream of TLRs on Kupffer cells (KCs) and hepatic stellate cells (HSCs) are poorly defined. Here, we describe the MAP3K tumor progression locus 2 (Tpl2) as being important for the activation of extracellular regulated kinase (ERK) signaling in KCs and HSCs responding to stimulation of TLR4 and TLR9. KCs lacking Tpl2 display defects with TLR induction of cytokines interleukin (IL)-1ß, IL-10, and IL-23. tpl2(-/-) HSCs were unable to increase expression of fibrogenic genes IL-1ß and tissue inhibitor of metalloproteinase 1 (TIMP-1), with the latter being the result of defective stimulation of TIMP-1 promoter activity by TLRs. To determine the in vivo relevance of Tpl2 signaling in liver fibrosis, we compared the fibrogenic responses of wild-type (WT) and tpl2(-/-) mice in three distinct models of chronic liver injury. In the carbon tetrachloride and methionine-choline-deficient diet models, we observed a significant reduction in fibrosis in mice lacking Tpl2, compared to WT controls. However, in the bile duct ligation model, there was no effect of tpl2 deletion, which may reflect a lesser role for HSCs in wounding response to biliary injury. CONCLUSION: We conclude that Tpl2 is an important signal transducer for TLR activation of gene expression in KCs and HSCs by the ERK pathway and that suppression of its catalytic activity may be a route toward suppressing fibrosis caused by hepatocellular injuries. (HEPATOLOGY 2013).
Assuntos
Células Estreladas do Fígado/fisiologia , Cirrose Hepática/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Animais , Células Cultivadas , Citocinas/metabolismo , Células Estreladas do Fígado/citologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Células de Kupffer/citologia , Células de Kupffer/fisiologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , MAP Quinase Quinase Quinases/genética , Macrófagos/citologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Transcrição Gênica/fisiologiaRESUMO
UNLABELLED: Phosphorylation of the RelA subunit at serine 536 (RelA-P-Ser536) is important for hepatic myofibroblast survival and is mechanistically implicated in liver fibrosis. Here, we show that a cell-permeable competing peptide (P6) functions as a specific targeted inhibitor of RelA-P-Ser536 in vivo and exerts an antifibrogenic effect in two progressive liver disease models, but does not impair hepatic inflammation or innate immune responses after lipopolysaccharide challenge. Using kinase assays and western blotting, we confirm that P6 is a substrate for the inhibitory kappa B kinases (IKKs), IKKα and IKKß, and, in human hepatic myofibroblasts, P6 prevents RelA-P-Ser536, but does not affect IKK activation of IκBα. We demonstrate that RelA-P-Ser536 is a feature of human lung and skin fibroblasts, but not lung epithelial cells, in vitro and is present in sclerotic skin and diseased lungs of patients suffering from idiopathic pulmonary fibrosis. CONCLUSION: RelA-P-Ser536 may be a core fibrogenic regulator of fibroblast phenotype.
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Imunidade Inata/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Adulto , Animais , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Fibroblastos/metabolismo , Humanos , Quinase I-kappa B/fisiologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Serina , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/farmacologiaRESUMO
Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease.
Assuntos
Cardiomegalia/etiologia , Miocárdio/patologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Angiotensinas/farmacologia , Animais , Pressão Sanguínea/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Fibrose , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/deficiência , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais/fisiologia , Remodelação Ventricular/fisiologiaRESUMO
Spinal Muscular Atrophy (SMA) is characterized by muscle atrophy and weakness and has an incidence of 1:11. 000 live births which projects an estimated population in the UK of 650-1,300 affected patients. Standards of Care (SoC) were updated in 2017 and they have been widely adopted as a reference for implementation of care in SMA across the globe. The effectiveness of implementation and adherence to these standards across different countries is unclear. The aim of this study is to describe the experience of individuals with SMA regarding their care in the UK. An online anonymised survey was sent out via patient organizations, the UK SMA Patient Registry, professional networks, and social media to reach across the UK. The survey captured demographic profile, professionals involved in a patient's care, Interventions and access to mobility aids and home adaptations. Participants responded about their access to services and to rate how important each professional and intervention was for their health and wellbeing. One hundred and twenty-eight responses were collected with a median age of 34 years (1-81). Seventy-three percent of participants were adults and 60% men. Overall good access to neurologist (>90%) but limited to nurse specialist (48%) and physiotherapist (57%). Good access to respiratory support was reported but limited for interventions for positioning and bracing and exercise. This survey highlights that access to certain professionals for people with SMA is limited in the UK. Striking differences were noted between pediatric and adult populations. Limited access to care were regularly reported, with half of the study population consistently not accessing full multidisciplinary care. Access to interventions for contracture management were recorded to have significant limitations. Mobility aids and home adaptations are widely available and were also reported as the most valued interventions. Access to nutritional support or speech and language therapy appears only to be available for a small proportion of the participants. Access to respiratory care was good especially in severe forms of SMA. We found pockets of good practice in the UK that align with the SoC. However, access is not equal for adults and children and access to certain professionals is significantly limited.
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Our primary aim was to establish the prevalence of pain within limb girdle muscular dystrophy R9 (LGMDR9). As part of the Global FKRP Registry, patients are asked to complete the Short Form McGill Pain Questionnaire (SF-MPQ) annually. We used the results of this questionnaire to determine individuals' maximum pain score and total pain score and examined overall pain intensity and associations between pain intensity and LGMDR9 genotypes, age, and ambulatory status. We also considered the pain descriptors used and pain progression over time. Of the 502 patients, 87% reported current pain and 25% reported severe current pain. We found no associations in pain severity between the different genotypes of LGMDR9. However, we did find statistically significant associations between pain severity and ambulatory status and between our paediatric and adult populations. We found pain descriptors to be more common words that one may associate with non-neural pain, and we found that a significant number of individuals (69%) reported a fluctuating pain pattern over time. We concluded that pain should be considered a significant issue among individuals with LGMDR9 requiring management. Implications regarding assessment of pain for other degenerative diseases are discussed.
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Exercise may ameliorate the eventual heart failure inherent in human aging. In this study, we use zebrafish to understand how aging and exercise affect cardiomyocyte turnover and myocardial remodelling. We show that cardiomyocyte proliferation remains constant throughout life but that onset of fibrosis is associated with a late increase in apoptosis. These findings correlate with decreases in voluntary swimming activity, critical swimming speed (Ucrit), and increases in biomarkers of cardiac insufficiency. The ability to respond to severe physiological stress is also impaired with age. Although young adult fish respond with robust cardiomyocyte proliferation in response to enforced swimming, this is dramatically impaired in older fish and served by a smaller proliferation-competent cardiomyocyte population. Finally, we show that these aging responses can be improved through increased activity throughout adulthood. However, despite improvement in Ucrit and the proliferative response to stress, the size of the proliferating cardiomyocyte population remained unchanged. The zebrafish heart models human aging and reveals the important trade-off between preserving cardiovascular fitness through exercise at the expense of accelerated fibrotic change.
Assuntos
Miócitos Cardíacos , Peixe-Zebra , Envelhecimento/fisiologia , Animais , Apoptose , Proliferação de Células , Coração/fisiologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: The Global FKRP Registry is a database for individuals with conditions caused by mutations in the Fukutin-Related Protein (FKRP) gene: limb girdle muscular dystrophy R9 (LGMDR9, formerly LGMD2I) and congenital muscular dystrophies MDC1C, Muscle-Eye-Brain Disease and Walker-Warburg Syndrome. The registry seeks to further understand the natural history and prevalence of FKRP-related conditions; aid the rapid identification of eligible patients for clinical studies; and provide a source of information to clinical and academic communities. METHODS: Registration is patient-initiated through a secure online portal. Data, reported by both patients and their clinicians, include: age of onset, presenting symptoms, family history, motor function and muscle strength, respiratory and cardiac function, medication, quality of life and pain. RESULTS: Of 663 registered participants, 305 were genetically confirmed LGMDR9 patients from 23 countries. A majority of LGMDR9 patients carried the common mutation c.826C > A on one or both alleles; 67.9% were homozygous and 28.5% were compound heterozygous for this mutation. The mean ages of symptom onset and disease diagnosis were higher in individuals homozygous for c.826C > A compared with individuals heterozygous for c.826C > A. This divergence was replicated in ages of loss of running ability, wheelchair-dependence and ventilation assistance; consistent with the milder phenotype associated with individuals homozygous for c.826C > A. In LGMDR9 patients, 75.1% were currently ambulant and 24.6%, nonambulant (unreported in 0.3%). Cardiac impairment was reported in 23.2% (30/129). INTERPRETATION: The Global FKRP Registry enables the collection of patient natural history data, which informs academics, healthcare professionals and industry. It represents a trial-ready cohort of individuals and is centrally placed to facilitate recruitment to clinical studies.
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Distrofia Muscular do Cíngulo dos Membros/genética , Pentosiltransferases/genética , Sistema de Registros , Síndrome de Walker-Warburg/genética , Adolescente , Adulto , Idade de Início , Idoso , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Fenótipo , Síndrome de Walker-Warburg/fisiopatologia , Adulto JovemRESUMO
PURPOSE: The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype. EXPERIMENTAL DESIGN: PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models. RESULTS: In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [3H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([3H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1). CONCLUSIONS: These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Colágeno Tipo I/fisiologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/metabolismo , Apoptose , Western Blotting , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Colágeno/química , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Pâncreas/citologia , Neoplasias Pancreáticas/metabolismo , Fenótipo , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT(2B)) on HSCs by serotonin, which activates expression of transforming growth factor ß1 (TGF-ß1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT(2B) enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT(2B) or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT(2B) attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT(2B) is clinically safe in humans and may be therapeutic in chronic liver disease.
Assuntos
Cirrose Hepática/terapia , Receptor 5-HT2B de Serotonina/metabolismo , Cicatrização , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Eletroforese em Gel de Poliacrilamida , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Imuno-Histoquímica , Indóis/farmacologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/metabolismo , Vírus Sinciciais Respiratórios/genética , Animais , Sítios de Ligação , Linhagem Celular , Proteínas do Nucleocapsídeo/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Transcrição GênicaRESUMO
Nucleocapsid (N) proteins from representative viruses of three genera within the Paramyxoviridae were expressed in insect cells using recombinant baculoviruses. RNA-containing structures, which appear morphologically identical to viral nucleocapsids, were isolated and subsequently imaged under a transmission electron microscope. Analysis of these images revealed marked differences in nucleocapsid morphology among the genera investigated, most notably between viruses of the Paramyxovirinae and the Pneumovirinae subfamilies. Helical pitch measurements were made, revealing that measles virus (MV, a Morbillivirus within the subfamily Paramyxovirinae) N protein produces helices that adopt multiple conformations with varying degrees of flexibility, while that of the Rubulavirus simian virus type 5 (SV5, subfamily Paramyxovirinae) produces more rigid structures with a less heterogeneous pitch distribution. Nucleocapsids produced by respiratory syncytial virus (RSV, subfamily Pneumovirinae) appear significantly narrower than those of MV and SV5 and have a longer pitch than the most extended form of MV. In addition to helical nucleocapsids, ring structures were also produced, image analysis of which has demonstrated that rings assembled from MV N protein consist of 13 subunits. This is consistent with previous reports that Sendai virus nucleocapsids have 13.07 subunits per turn. It was determined, however, that SV5 subnucleocapsid rings have 14 subunits, while rings derived from the radically different RSV nucleocapsid have been found to contain predominantly 10 subunits.