RESUMEN
Intermediate filaments (IFs) are cytoskeletal and nucleoskeletal structures that provide mechanical and stress-coping resilience to cells, contribute to subcellular and tissue-specific biological functions, and facilitate intracellular communication. IFs, including nuclear lamins and those in the cytoplasm (keratins, vimentin, desmin, neurofilaments and glial fibrillary acidic protein, among others), are functionally regulated by post-translational modifications (PTMs). Proteomic advances highlight the enormous complexity and regulatory potential of IF protein PTMs, which include phosphorylation, glycosylation, sumoylation, acetylation and prenylation, with novel modifications becoming increasingly appreciated. Future studies will need to characterize their on-off mechanisms, crosstalk and utility as biomarkers and targets for diseases involving the IF cytoskeleton.
Asunto(s)
Proteínas de Filamentos Intermediarios/metabolismo , Modelos Biológicos , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND AND AIMS: Hepatocyte keratin polypeptides 8/18 (K8/K18) are unique among intermediate filaments proteins (IFs) in that their mutation predisposes to, rather than causes, human disease. Mice that overexpress human K18 R90C manifest disrupted hepatocyte keratin filaments with hyperphosphorylated keratins and predisposition to Fas-induced liver injury. We hypothesized that high-throughput screening will identify compounds that protect the liver from mutation-triggered predisposition to injury. APPROACH AND RESULTS: Using A549 cells transduced with a lentivirus K18 construct and high-throughput screening, we identified the SRC-family tyrosine kinases inhibitor, PP2, as a compound that reverses keratin filament disruption and protects from apoptotic cell death caused by K18 R90C mutation at this highly conserved arginine. PP2 also ameliorated Fas-induced apoptosis and liver injury in male but not female K18 R90C mice. The PP2 male selectivity is due to its lower turnover in male versus female livers. Knockdown of SRC but not another kinase target of PP2, protein tyrosine kinase 6, in A549 cells abrogated the hepatoprotective effect of PP2. Phosphoproteomic analysis and validation showed that the protective effect of PP2 associates with Ser/Thr but not Tyr keratin hypophosphorylation, and differs from the sex-independent effect of the Ser/Thr kinase inhibitor PKC412. Inhibition of RAF kinase, a downstream target of SRC, by vemurafenib had a similar protective effect to PP2 in A549 cells and male K18 R90C mice. CONCLUSIONS: PP2 protects, in a male-selective manner, keratin mutation-induced mouse liver injury by inhibiting SRC-triggered downstream Ser/Thr phosphorylation of K8/K18, which is phenocopied by RAF kinase inhibitor vemurafenib. The PP2/vemurafenib-associated findings, and their unique mechanisms of action, further support the potential role of select kinase inhibition as therapeutic opportunities for keratin and other IF-associated human diseases.
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Queratinas , Familia-src Quinasas , Ratones , Masculino , Humanos , Animales , Queratinas/metabolismo , Familia-src Quinasas/metabolismo , Vemurafenib/metabolismo , Vemurafenib/farmacología , Ratones Transgénicos , Hígado/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Mutación , Queratina-18RESUMEN
Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.
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Hiponatremia , Hepatopatías , Animales , Ratones , Carbamoil Fosfato/metabolismo , Ornitina Carbamoiltransferasa/metabolismo , Citocromos c/metabolismo , Hiponatremia/metabolismo , Hiponatremia/patología , Hepatocitos/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Hepatopatías/metabolismo , Mitocondrias/metabolismo , Urea/metabolismoRESUMEN
There is increasing evidence for the importance of the nuclear envelope in lipid metabolism, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Human mutations in LMNA, encoding A-type nuclear lamins, cause early-onset insulin resistance and NASH, while hepatocyte-specific deletion of Lmna predisposes to NASH with fibrosis in male mice. Given that variants in the gene encoding LAP2α, a nuclear protein that regulates lamin A/C, were previously identified in patients with NAFLD, we sought to determine the role of LAP2α in NAFLD using a mouse genetic model. Hepatocyte-specific Lap2α-knockout (Lap2α(ΔHep)) mice and littermate controls were fed normal chow or high-fat diet (HFD) for 8 wk or 6 mo. Unexpectedly, male Lap2α(ΔHep) mice showed no increase in hepatic steatosis or NASH compared with controls. Rather, Lap2α(ΔHep) mice demonstrated reduced hepatic steatosis, with decreased NASH and fibrosis after long-term HFD. Accordingly, pro-steatotic genes including Cidea, Mogat1, and Cd36 were downregulated in Lap2α(ΔHep) mice, along with concomitant decreases in expression of pro-inflammatory and pro-fibrotic genes. These data indicate that hepatocyte-specific Lap2α deletion protects against hepatic steatosis and NASH in mice and raise the possibility that LAP2α could become a potential therapeutic target in human NASH.NEW & NOTEWORTHY The nuclear envelope and lamina regulate lipid metabolism and susceptibility to nonalcoholic steatohepatitis (NASH), but the role of the nuclear lamin-binding protein LAP2α in NASH has not been explored. Our data demonstrate that hepatocyte-specific loss of LAP2α protects against diet-induced hepatic steatosis, NASH, and fibrosis in male mice, with downregulation of pro-steatotic, pro-inflammatory, and pro-fibrotic lamin-regulated genes. These findings suggest that targeting LAP2α could have future potential as a novel therapeutic avenue in NASH.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Masculino , Ratones , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Laminas/metabolismo , Hígado/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/prevención & control , Cirrosis Hepática/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/prevención & controlRESUMEN
BACKGROUND & AIMS: Carbamoyl phosphate synthetase 1 (CPS1) is a highly abundant mitochondrial urea cycle enzyme that is expressed primarily in hepatocytes. CPS1 is constitutively and physiologically secreted into bile but is released into the bloodstream upon acute liver injury (ALI). Given its abundance and known short half-life, we tested the hypothesis that it may serve as a prognostic serum biomarker in the setting of acute liver failure (ALF). METHODS: CPS1 levels were determined using enzyme-linked immunosorbent assay and immunoblotting of sera collected by the ALF Study Group (ALFSG) from patients with ALI and ALF (103 patients with acetaminophen and 167 non-acetaminophen ALF etiologies). A total of 764 serum samples were examined. The inclusion of CPS1 was compared with the original ALFSG Prognostic Index by area under the receiver operating characteristic curve analysis. RESULTS: CPS1 values for acetaminophen-related patients were significantly higher than for non-acetaminophen patients (P < .0001). Acetaminophen-related patients who received a liver transplant or died within 21 days of hospitalization exhibited higher CPS1 levels than patients who spontaneously survived (P = .01). Logistic regression and area under the receiver operating characteristic analysis of CPS1 enzyme-linked immunosorbent assay values improved the accuracy of the ALFSG Prognostic Index, which performed better than the Model for End-Stage Liver Disease, in predicting 21-day transplant-free survival for acetaminophen- but not non-acetaminophen-related ALF. An increase of CPS1 but not alanine transaminase or aspartate transaminase, when comparing day 3 with day 1 levels was found in a higher percentage of acetaminophen transplanted/dead patients (P < .05). CONCLUSION: Serum CPS1 determination provides a new potential prognostic biomarker to assess patients with acetaminophen-induced ALF.
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Enfermedad Hepática en Estado Terminal , Fallo Hepático Agudo , Humanos , Acetaminofén/efectos adversos , Biomarcadores , Carbamoil Fosfato , Ligasas , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/diagnóstico , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and are associated with the accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about the cytotoxic effects of these porphyrin nanostructures. Previously, we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), to cause organelle-selective protein aggregation, which we posited to be a major mechanism by which fluorescent porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein-binding ability. UV-visible spectrophotometry showed pH-mediated reversible transformations of PP-IX nanostructures. Biochemical analysis showed that PP-IX nanostructure size modulated PP-IX-induced protein oxidation and protein aggregation. Furthermore, albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. PP-IX binding quenched albumin intrinsic fluorescence and oxidized His-91 residue to Asn/Asp, likely via a previously described photo-oxidation mechanism for other proteins. Extracellular albumin protected from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX nanostructures in the context of porphyrias and offers insights into potential novel therapeutic approaches.
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Nanoestructuras/química , Agregado de Proteínas , Protoporfirinas/química , Ácido Aminolevulínico/farmacología , Animales , Sitios de Unión , Bovinos , Línea Celular , Deferoxamina/farmacología , Dimerización , Humanos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Oxidación-Reducción , Unión Proteica , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Aberrant T cell development is a pivotal risk factor for autoimmune disease; however, the underlying molecular mechanism of T cell overactivation is poorly understood. Here, we identified NF-κB-inducing kinase (NIK) and IkB kinase α (IKKα) in thymic epithelial cells (TECs) as essential regulators of T cell development. Mouse TEC-specific ablation of either NIK or IKKα resulted in severe T cell-mediated inflammation, injury, and fibrosis in the liver and lung, leading to premature death within 18 d of age. NIK or IKKα deficiency abrogated medullary TEC development, and led to breakdown of central tolerance, production of autoreactive T cells, and fatal autoimmune destruction in the liver and lung. TEC-specific ablation of NIK or IKKα also impaired thymic T cell development from the double-negative through the double-positive stages and inhibited peripheral B cell development. These results unravel a hitherto unrecognized essential role of TEC-intrinsic NIK and IKKα pathways in autoimmunity and T cell-instigated chronic liver and lung diseases.
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Autoinmunidad/inmunología , Quinasa I-kappa B/fisiología , Inflamación/inmunología , Hígado/inmunología , Pulmón/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Diferenciación Celular , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Homeostasis , Inflamación/metabolismo , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Timo/metabolismo , Timo/patología , Quinasa de Factor Nuclear kappa BRESUMEN
Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury.
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Lesión Pulmonar Aguda/metabolismo , Ácidos y Sales Biliares/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Acetaminofén/metabolismo , Lesión Pulmonar Aguda/enzimología , Adulto , Animales , Bilis/metabolismo , Citocinas/metabolismo , Proteína Ligando Fas/metabolismo , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hepatopatías , Macrófagos/metabolismo , Masculino , Ratones , Mitocondrias/metabolismoRESUMEN
Laminopathies are rare diseases associated with mutations in LMNA, which encodes nuclear lamin A/C. LMNA variants lead to diverse tissue-specific phenotypes including cardiomyopathy, lipodystrophy, myopathy, neuropathy, progeria, bone/skin disorders, and overlap syndromes. The mechanisms underlying these heterogeneous phenotypes remain poorly understood, although post-translational modifications, including phosphorylation, are postulated as regulators of lamin function. We catalogued all known lamin A/C human mutations and their associated phenotypes, and systematically examined the putative role of phosphorylation in laminopathies. In silico prediction of specific LMNA mutant-driven changes to lamin A phosphorylation and protein structure was performed using machine learning methods. Some of the predictions we generated were validated via assessment of ectopically expressed wild-type and mutant LMNA. Our findings indicate phenotype- and mutant-specific alterations in lamin phosphorylation, and that some changes in phosphorylation may occur independently of predicted changes in lamin protein structure. Therefore, therapeutic targeting of phosphorylation in the context of laminopathies will likely require mutant- and kinase-specific approaches.
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Estudios de Asociación Genética , Genotipo , Lamina Tipo A/genética , Laminopatías/patología , Mutación , Fenotipo , Femenino , Humanos , Lamina Tipo A/metabolismo , Laminopatías/clasificación , Laminopatías/genética , Laminopatías/metabolismo , Masculino , FosforilaciónRESUMEN
Climate change has been described as the greatest public health threat of the 21st century. It has significant implications for digestive health. A multinational team with representation from all continents, excluding Antarctica and covering 18 countries, has formulated a commentary which outlines both the implications for digestive health and ways in which this challenge can be faced.
Asunto(s)
Cambio Climático , Gastroenterología , HumanosRESUMEN
Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 ß-cells is dependent on the presence and levels of K18. K18-knockout (K18â/â) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8â/â islets. K7, akin to F-actin, is concentrated at the apical vertex of ß-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8â/â ß-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse ß-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.
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Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/patología , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Queratina-18/genética , Queratina-7/genética , Queratina-8/genética , Ratones , Ratones Noqueados , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/ß-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury. METHODS: ß-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2â¯weeks. ß-Catenin was exogenously inhibited in hepatocytes by administering ß-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4â¯weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry. RESULTS: DDC-fed mice lacking ß-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent ß-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking ß-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of ß-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking ß-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury. CONCLUSIONS: ß-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria. LAY SUMMARY: Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/ß-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting ß-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting ß-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.
Asunto(s)
Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Porfirias/complicaciones , Porfirinas/metabolismo , beta Catenina/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Hepatocitos/patología , Inmunohistoquímica , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones NoqueadosRESUMEN
The nuclear lamina is a multi-protein lattice composed of A- and B-type lamins and their associated proteins. This protein lattice associates with heterochromatin and integral inner nuclear membrane proteins, providing links among the genome, nucleoskeleton, and cytoskeleton. In the 1990s, mutations in EMD and LMNA were linked to Emery-Dreifuss muscular dystrophy. Since then, the number of diseases attributed to nuclear lamina defects, including laminopathies and other disorders, has increased to include more than 20 distinct genetic syndromes. Studies of patients and mouse genetic models have pointed to important roles for lamins and their associated proteins in the function of gastrointestinal organs, including liver and pancreas. We review the interactions and functions of the lamina in relation to the nuclear envelope and genome, the ways in which its dysfunction is thought to contribute to human disease, and possible avenues for targeted therapies.
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Enfermedades Gastrointestinales/genética , Laminas/fisiología , Lámina Nuclear/fisiología , Animales , Citoesqueleto/metabolismo , Genoma , Humanos , Laminas/química , Hígado/citología , Ratones , Páncreas/citologíaRESUMEN
We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain-luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell-specific disruption of Hif1a (Hif1aAc-/-) developed spontaneous endoplasmic reticulum stress and less severe AP, but did not accumulate Fib-γD following administration of cerulein. Feeding mice increased pancreatic expression of HIF1α, indicating a physiologic role in the exocrine pancreas. Therefore, HIF1α has bifunctional roles, in exocrine pancreas homeostasis and progression of AP that is promoted by intrapancreatic coagulation.
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Células Acinares/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Páncreas/citología , Pancreatitis/genética , Enfermedad Aguda , Animales , Arginina , Ceruletida , Modelos Animales de Enfermedad , Homeostasis/genética , Humanos , Ratones , Páncreas Exocrino/metabolismo , Pancreatitis/inducido químicamente , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Lamins have important roles in nuclear structure and cell signaling. Several diseases are associated with mutations in the lamin A/C gene (LMNA in humans). Patients with familial partial lipodystrophy caused by LMNA mutations develop pancreatitis, but lamin function in the pancreas and how these mutations affect pancreatic regulation are unknown. We generated mice with inducible exocrine pancreas-specific disruption of Lmna and showed that LMNA is lost from most exocrine pancreas cells. LMNA-knockout pancreata develop endoplasmic reticulum stress with loss of acinar cell markers, increased autophagy, apoptosis, and cell proliferation, compared to CreERT2- mice (littermate controls). Disruption of Lmna led to a phenotype that resembled chronic pancreatitis, with increased Sirius Red staining and α-smooth muscle actin in male LMNA-knockout mice compared to littermate males, but not in female mice. LMNA-knockout pancreata have reduced levels of RB and activation of E2F, based on increased expression of E2F target genes. Therefore, lamins maintain pancreatic homeostasis by regulating RB stability and E2F activity.
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Factores de Transcripción E2F/fisiología , Homeostasis/genética , Lamina Tipo A/fisiología , Páncreas Exocrino/metabolismo , Proteína de Retinoblastoma/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genéticaRESUMEN
Alcohol liver disease (ALD) is one of the major chronic liver diseases worldwide, ranging from fatty liver, alcoholic hepatitis, cirrhosis, and potentially, hepatocellular carcinoma. Epidemiological studies suggest a potential link between ALD and impaired circadian rhythms, but the role of hepatic circadian proteins in the pathogenesis of ALD remains unknown. Here we show that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect mice from ALD. Ethanol diet-fed mice with liver-specific knockout (Bmal1-LKO) or depletion of Bmal1 develop more severe liver steatosis and injury as well as a simultaneous suppression of both de novo lipogenesis and fatty acid oxidation, which can be rescued by the supplementation of synthetic PPARα ligands. Restoring de novo lipogenesis in the liver of Bmal1-LKO mice by constitutively active AKT not only elevates hepatic fatty acid oxidation but also alleviates ethanol-induced fatty liver and liver injury. Furthermore, hepatic over-expression of lipogenic transcription factor ChREBP, but not SREBP-1c, in the liver of Bmal1-LKO mice also increases fatty acid oxidation and partially reduces ethanol-induced fatty liver and liver injury. Conclusion: we identified a protective role of BMAL1 in hepatocytes against ALD. The protective action of BMAL1 during alcohol consumption depends on its ability to couple ChREBP-induced de novo lipogenesis with PPARα-mediated fatty oxidation. (Hepatology 2018).
Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Hepatocitos/metabolismo , Lipogénesis , Hepatopatías Alcohólicas/metabolismo , PPAR alfa/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Femenino , Hepatopatías Alcohólicas/etiología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina-associated proteins predispose to NAFLD and used a candidate gene-sequencing approach to test for variants in 10 nuclear lamina-related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina-related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina-associated polypeptide-2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin-LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin-binding protein p62/SQSTM1. CONCLUSION: Several variants in nuclear lamina-related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710-1725).