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1.
Hepatology ; 45(1): 88-96, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17187412

RESUMO

UNLABELLED: Several human liver diseases are associated with formation of Mallory body (MB) inclusions. These hepatocyte cytoplasmic deposits are composed primarily of hyperphosphorylated keratins 8 and 18 (K8/K18). Feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet is a well-established mouse model of MBs. K8 overexpression, and K8-null or K18-null mouse models, indicate that a K8-greater-than-K18 expression ratio is critical for MB formation. We used established transgenic mouse models to study the effect of K18 overexpression and phosphorylation, or keratin filament disorganization, on MB formation. Five mouse lines were used: nontransgenic, those that overexpress wild-type K18 or the K18 phosphorylation mutants Ser33-to-Ala (S33A) or Ser52-to-Ala (S52A), and mice that overexpress K18 Arg89-to-Cys, which causes collapse of the keratin filament network into dots. DDC feeding induced MBs in nontransgenic livers, but MBs were rarely seen in any of the K18 transgenic mice. Wild-type K18 overexpression protected mice from DDC-induced liver injury. CONCLUSION: K18 overexpression protects mice from MB formation and from DDC-induced liver injury, which supports the importance of the K8-to-K18 ratio in MB formation. The effect of K18 on MB formation is independent of hepatocyte keratin filament organization or K18 Ser33/Ser52 phosphorylation. Keratin filament collapse, which is a major risk for acute liver injury, is well tolerated in the context of chronic DDC-mediated liver injury.


Assuntos
Hepatócitos/metabolismo , Corpos de Inclusão/metabolismo , Filamentos Intermediários/metabolismo , Queratina-18/metabolismo , Fígado/patologia , Animais , Dicarbetoxi-Di-Hidrocolidina , Feminino , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/ultraestrutura , Corpos de Inclusão/ultraestrutura , Filamentos Intermediários/ultraestrutura , Queratina-18/genética , Queratina-8/genética , Queratina-8/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Fosforilação
2.
Hepatology ; 40(2): 459-66, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15368451

RESUMO

Keratin 8 and 18 (K8/18) phosphorylation plays a significant and site-specific role in regulating keratin filament organization, association with binding proteins, and modulation of cell cycle progression. Keratin hyperphosphorylation correlates with exposure to a variety of stresses in cultured cells and in mouse models of liver, pancreatic, and gallbladder injury, and it is found in association with mouse and human Mallory bodies. We asked whether K8/18 phosphorylation correlates with human liver disease progression by analyzing liver explants and biopsies of patients with chronic noncirrhotic hepatitis C virus (HCV) or cirrhosis. We also examined the effect of HCV therapy with interleukin-10 on keratin phosphorylation. Using site-specific antiphosphokeratin antibodies we found keratin hyperphosphorylation on most K8/18 sites in all cirrhotic liver explants tested and in most liver biopsies from patients with chronic HCV infection. Immunofluorescence staining of precirrhotic HCV livers showed focal keratin hyperphosphorylation and limited reorganization of keratin filament networks. In cirrhotic livers, keratin hyperphosphorylation occurred preferentially in hepatic nodule cells adjacent to bridging fibrosis and associated with increased stress kinase activation and apoptosis. Histological and serological improvement after interleukin-10 therapy was accompanied by normalization of keratin hyperphosphorylation on some sites in 7 of 10 patients. In conclusion, site-specific keratin phosphorylation in liver disease is a progression marker when increased and a likely regression marker when decreased.


Assuntos
Hepatite C Crônica/fisiopatologia , Queratinas/metabolismo , Cirrose Hepática/fisiopatologia , Biomarcadores/análise , Estudos de Casos e Controles , Progressão da Doença , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Hepatócitos/metabolismo , Humanos , Interleucina-10/farmacologia , Queratina-18 , Queratina-8 , Queratinas/genética , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Mutação , Fosforilação/efeitos dos fármacos
3.
J Cell Sci ; 117(Pt 9): 1709-19, 2004 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15075232

RESUMO

Keratin polypeptides 8 and 18 (K8/K18) are the major intermediate filament proteins of pancreatic acinar cells and hepatocytes. Pancreatic keratin function is unknown, whereas hepatocyte keratins protect from mechanical and non-mechanical forms of stress. We characterized steady-state pancreatic keratin expression in Balb/c mice after caerulein and choline-deficient ethionine-supplemented diet (CDD), or on exposure to the generalized stresses of heat and water immersion. Keratins were studied at the protein, RNA and organizational levels. Isolated acini were used to study the role of nuclear factor (NF)-kappaB using selective inhibitors. Keratins were found to be abundant proteins making up 0.2%, 0.3% and 0.5% of the total cellular protein of pancreas, liver and small intestine, respectively. Caerulein and CDD caused a threefold transcription-mediated overall increase in K8/K18/K19/K20 proteins. Keratin overexpression begins on tissue recovery, peaks 2 days after caerulein injection, or 1 day after CDD discontinuation, and returns to basal levels after 10 days. K19/K20-containing cytoplasmic filaments are nearly absent pre-injury but form post-injury then return to their original membrane-proximal distribution after 10 days. By contrast, generalized stresses of heat or water-immersion stress do not alter keratin expression levels. Caerulein-induced keratin overexpression is associated with NF-kappaB activation when tested using ex vivo acinar cell cultures. In conclusion, keratins are abundant proteins that can behave as stress proteins in response to tissue-specific but not generalized forms of injury. Pancreatic keratin overexpression is associated with NF-kappaB activation and may serve unique functions in acinar or ductal cell response to injury.


Assuntos
Regulação da Expressão Gênica , Temperatura Alta , Queratinas/genética , Queratinas/metabolismo , NF-kappa B/metabolismo , Pâncreas/metabolismo , Animais , Ceruletídeo/farmacologia , Deficiência de Colina , Dieta , Etionina/farmacologia , Febre/genética , Febre/metabolismo , Intestino Delgado/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Pâncreas/efeitos dos fármacos , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologia , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Água/farmacologia
4.
J Cell Sci ; 116(Pt 22): 4629-38, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14576356

RESUMO

Keratin transgenic mouse models and the association of human keratin mutations with liver disease highlight the importance of keratins in protecting the liver from environmental insults, but little is known regarding keratins and their function in the gallbladder. We characterized keratin expression pattern and filament organization in normal and keratin polypeptide-8 (K8)-null, K18-null and K19-null gallbladders, and examined susceptibility to liver and gallbladder injury induced by a high-fat lithogenic diet (LD) in K8-null mice. The major keratins of normal mouse gallbladder are K8>K19>K18 which become markedly depleted in K8-null mice with minor K18/K19 remnants and limited K7 over-expression. Compensatory K18/K20 protein and RNA overexpression occur in K19-null but not in K18-null gallbladders, probably because of the higher levels of K19 than K18 in normal gallbladder. LD challenge causes more severe liver injury in K8-null than wild-type mice without altering keratin protein levels. In contrast, wild-type and K8-null gallbladders are equally susceptible to LD-induced injury and stone formation, but wild-type gallbladders do overexpress keratins upon LD challenge. LD-induced injury triggers keratin hyperphosphorylation in wild-type livers and gallbladders. Hence, mouse gallbladder K8/K18/K19 expression is induced in response to cholelithiasis injury. A high-fat LD increases the susceptibility of K8-null mice to liver but not gallbladder injury, which suggests that keratin mutations may increase the risk of liver damage in patients with steatohepatitis. Differences between K8-null mouse gallbladder and hepatocyte susceptibility to injury may be related to their minimal versus absent keratin expression, respectively.


Assuntos
Dieta , Vesícula Biliar/metabolismo , Queratinas/metabolismo , Fígado/metabolismo , Animais , Suscetibilidade a Doenças/patologia , Técnica Indireta de Fluorescência para Anticorpo , Vesícula Biliar/patologia , Queratina-8 , Queratinas/genética , Fígado/patologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Proc Natl Acad Sci U S A ; 99(7): 4373-8, 2002 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-11917136

RESUMO

Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33-->Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3zeta mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia in vivo. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.


Assuntos
Hepatócitos/fisiologia , Queratinas/fisiologia , Mitose , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Animais , Hepatectomia , Queratinas/química , Queratinas/genética , Camundongos , Camundongos Transgênicos , Mutação , Fosforilação , Tirosina 3-Mono-Oxigenase/análise
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