RESUMO
Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.
Assuntos
Autofagia , Ceruletídeo/toxicidade , Pancreatite/patologia , Tripsina/metabolismo , Tripsinogênio/metabolismo , Animais , Autofagossomos/metabolismo , Endossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Vesículas Secretórias/metabolismoRESUMO
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Neoplasias Pancreáticas/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/prevenção & controle , Carcinoma Ductal Pancreático/secundário , Catepsinas/genética , Catepsinas/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genes ras , Heterozigoto , Homozigoto , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Transdução de Sinais , Carga Tumoral , Células Tumorais CultivadasRESUMO
BACKGROUND: Chemoresistance is a main obstacle to effective esophageal cancer (EC) therapy. We hypothesize that altered expression of microRNAs (miRNAs) play a role in EC cancer progression and resistance to 5-fluorouracil (5-FU) based chemotherapeutic strategies. METHODS: Four pairs of esophageal adenocarcinoma (EAC) cell lines and corresponding 5-FU resistant variants were established. The expression levels of miRNAs previously shown to be involved in the general regulation of stem cell pathways were analyzed by qRT-PCR. The effects of selected miRNAs on proliferation, apoptosis, and chemosensitivity were evaluated both in vitro and in vivo. We identified a particular miRNA and analyzed its putative target genes in 14 pairs of human EC tumor specimens with surrounding normal tissue by qRT-PCR as well as Wnt pathway associated genes by immunohistochemistry in another 45 EAC tumor samples. RESULTS: MiR-221 was overexpressed in 5-FU resistant EC cell lines as well as in human EAC tissue. DKK2 was identified as a target gene for miR-221. Knockdown of miR-221 in 5-FU resistant cells resulted in reduced cell proliferation, increased apoptosis, restored chemosensitivity, and led to inactivation of the Wnt/ß-catenin pathway mediated by alteration in DKK2 expression. Moreover, miR-221 reduction resulted in alteration of EMT-associated genes such as E-cadherin and vimentin as well as significantly slower xenograft tumor growth in nude mice. RT profiler analysis identified a substantial dysregulation of 4 Wnt/ß-catenin signaling and chemoresistance target genes as a result of miR-221 modulation: CDH1, CD44, MYC, and ABCG2. CONCLUSION: MiR-221 controls 5-FU resistance of EC partly via modulation of Wnt/ß-catenin-EMT pathways by direct targeting of DKK2 expression. MiR-221 may serve as a prognostic marker and therapeutic target for patients with 5-FU resistant EAC.
Assuntos
Adenocarcinoma/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/metabolismo , Fluoruracila/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , MicroRNAs/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.
Assuntos
Cisteína Endopeptidases/metabolismo , Macrófagos/enzimologia , Pancreatite/enzimologia , Animais , Ceruletídeo/toxicidade , Cisteína Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancreatite/induzido quimicamenteRESUMO
Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.
Assuntos
Apoptose , Catepsina B/metabolismo , Pâncreas/enzimologia , Pancreatite/patologia , Peptídeo Hidrolases/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancreatite/enzimologia , Frações Subcelulares/enzimologiaRESUMO
BACKGROUND & AIMS: Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice. METHODS: Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3(-/-) mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2(-/-) mice, which develop spontaneous biliary inflammation, as well as Bcl3(-/-)Mdr2(-/-) mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3(-/-) mice were irradiated, injected with bone marrow from Bcl3(-/-) or control mice, and AP was induced. RESULTS: Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2(-/-) mice than from control mice. Bcl3(-/-) mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3(-/-) mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3(-/-) mice with AP, compared with control mice with AP. Bcl3(-/-)Mdr2(-/-) mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2(-/-) mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammation during development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA. CONCLUSIONS: BCL3 is up-regulated in inflamed pancreatic or biliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.
Assuntos
Ductos Biliares/metabolismo , Colangite Esclerosante/prevenção & controle , Pâncreas/metabolismo , Pancreatite/prevenção & controle , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Doença Aguda , Animais , Proteína 3 do Linfoma de Células B , Ductos Biliares/patologia , Transplante de Medula Óssea , Ceruletídeo , Colangite Esclerosante/genética , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Humanos , Proteínas I-kappa B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , Subunidade p50 de NF-kappa B/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Proteólise , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Ácido Taurocólico , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ubiquitinação , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND & AIMS: Little is known about the pathogenic mechanisms of chronic pancreatitis. We investigated the roles of complement component 5 (C5) in pancreatic fibrogenesis in mice and patients. METHODS: Chronic pancreatitis was induced by ligation of the midpancreatic duct, followed by a single supramaximal intraperitoneal injection of cerulein, in C57Bl6 (control) and C5-deficient mice. Some mice were given injections of 2 different antagonists of the receptor for C5a over 21 days. In a separate model, mice were given injections of cerulein for 10 weeks to induce chronic pancreatitis. Direct effects of C5 were studied in cultured primary cells. We performed genotype analysis for the single-nucleotide polymorphisms rs 17611 and rs 2300929 in C5 in patients with pancreatitis and healthy individuals (controls). Blood cells from 976 subjects were analyzed by transcriptional profiling. RESULTS: During the initial phase of pancreatitis, levels of pancreatic damage were similar between C5-deficient and control mice. During later stages of pancreatitis, C5-deficient mice and mice given injections of C5a-receptor antagonists developed significantly less pancreatic fibrosis than control mice. Primary pancreatic stellate cells were activated in vitro by C5a. There were no differences in the rs 2300929 SNP between subjects with or without pancreatitis, but the minor allele rs17611 was associated with a significant increase in levels of C5 in whole blood. CONCLUSIONS: In mice, loss of C5 or injection of a C5a-receptor antagonist significantly reduced the level of fibrosis of chronic pancreatitis, but this was not a consequence of milder disease in early stages of pancreatitis. C5 might be a therapeutic target for chronic pancreatitis.
Assuntos
Complemento C5/metabolismo , Células Estreladas do Pâncreas/metabolismo , Pancreatite Crônica/metabolismo , Compostos de Anilina/farmacologia , Animais , Estudos de Casos e Controles , Ceruletídeo , Complemento C5/deficiência , Complemento C5/genética , Modelos Animais de Doenças , Fibrose , Predisposição Genética para Doença , Ligadura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ductos Pancreáticos/cirurgia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/imunologia , Células Estreladas do Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/tratamento farmacológico , Pancreatite Crônica/genética , Pancreatite Crônica/imunologia , Pancreatite Crônica/patologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/metabolismo , Tetra-Hidronaftalenos/farmacologia , Fatores de TempoRESUMO
Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.
Assuntos
Cardiolipinas/metabolismo , Citrato (si)-Sintase/metabolismo , Isquemia/metabolismo , Fígado/metabolismo , Animais , Isquemia/patologia , Lipogênese , Fígado/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosfolipídeos/metabolismo , Ratos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND & AIMS: Little is known about the mechanisms of the progressive tissue destruction, inflammation, and fibrosis that occur during development of chronic pancreatitis. Autophagy is involved in multiple degenerative and inflammatory diseases, including pancreatitis, and requires the protein autophagy related 5 (ATG5). We created mice with defects in autophagy to determine its role in pancreatitis. METHODS: We created mice with pancreas-specific disruption of Atg5 (Ptf1aCreex1;Atg5F/F mice) and compared them to control mice. Pancreata were collected and histology, immunohistochemistry, transcriptome, and metabolome analyses were performed. ATG5-deficient mice were placed on diets containing 25% palm oil and compared with those on a standard diet. Another set of mice received the antioxidant N-acetylcysteine. Pancreatic tissues were collected from 8 patients with chronic pancreatitis (CP) and compared with pancreata from ATG5-deficient mice. RESULTS: Mice with pancreas-specific disruption of Atg5 developed atrophic CP, independent of ß-cell function; a greater proportion of male mice developed CP than female mice. Pancreata from ATG5-deficient mice had signs of inflammation, necrosis, acinar-to-ductal metaplasia, and acinar-cell hypertrophy; this led to tissue atrophy and degeneration. Based on transcriptome and metabolome analyses, ATG5-deficient mice produced higher levels of reactive oxygen species than control mice, and had insufficient activation of glutamate-dependent metabolism. Pancreata from these mice had reduced autophagy, increased levels of p62, and increases in endoplasmic reticulum stress and mitochondrial damage, compared with tissues from control mice; p62 signaling to Nqo1 and p53 was also activated. Dietary antioxidants, especially in combination with palm oil-derived fatty acids, blocked progression to CP and pancreatic acinar atrophy. Tissues from patients with CP had many histologic similarities to those from ATG5-deficient mice. CONCLUSIONS: Mice with pancreas-specific disruption of Atg5 develop a form of CP similar to that of humans. CP development appears to involve defects in autophagy, glutamate-dependent metabolism, and increased production of reactive oxygen species. These mice might be used to identify therapeutic targets for CP.
Assuntos
Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Associadas aos Microtúbulos/genética , Pâncreas/metabolismo , Pancreatite Crônica/genética , Acetilcisteína/farmacologia , Animais , Atrofia , Autofagia/imunologia , Proteína 5 Relacionada à Autofagia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/imunologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Inflamação , Masculino , Camundongos , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/metabolismo , Óleo de Palmeira , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Pancreatite Crônica/imunologia , Pancreatite Crônica/patologia , Óleos de Plantas/farmacologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Fatores Sexuais , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.
Assuntos
Quimotripsina/metabolismo , Tripsinogênio/química , Tripsinogênio/metabolismo , Sequência de Aminoácidos , Animais , Cátions , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Ativação Enzimática , Genoma/genética , Humanos , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Pâncreas/enzimologia , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Tripsinogênio/genéticaRESUMO
OBJECTIVES: C-telopeptide crosslaps (CTX) and bone-specific alkaline phosphatase (BAP) do not provide sufficient sensitivity and specificity for diagnosis of osteoporosis. Cathepsin K (CatK), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (total (t) and soluble (s) RANKL) play an important role in bone metabolism. Thus serum levels of biochemical markers, each or in combination, may be useful in diagnosis of osteoporosis. STUDY DESIGN: In total, 121 healthy women, 27 premenopausal women aged between 20 and 45 years, and 94 postmenopausal women aged 59-81 years, all free of known skeletal disorders were included. They underwent bone density measurement and measurement of biochemical markers. MAIN OUTCOME MEASURES: Based on WHO criteria, women were stratified in four groups (premenopausal: healthy; postmenopausal: healthy, osteopenia, osteoporosis), and their levels of CatK, OPG, RANKL, CTX and BAP were analyzed. RESULTS: Using WHO criteria 21 postmenopausal women had normal bone mineral density (BMD), 49 had osteopenia and 24 had osteoporosis. There were no significant correlations of CatK, OPG and RANKL with BMD (T-score) in age-adjusted analysis, but for BAP and CTX. ROC analyses resulted in poor diagnostic validity of all parameters. The best result - also confirmed by discriminant analysis - was yielded by BAP (AUC=0.646 [0.510; 0.781]). A combination of variables did not significantly improve the diagnostic power. CONCLUSIONS: Baseline serum levels of BAP, CTX, CatK, OPG, sRANKL or tRANKL alone or in combination are not suitable to distinguish osteoporotic from non-osteoporotic postmenopausal women with sufficient accuracy.
Assuntos
Osso e Ossos/enzimologia , Catepsina K/sangue , Colágeno Tipo I/sangue , Osteoporose/sangue , Osteoporose/diagnóstico , Osteoprotegerina/sangue , Peptídeos/sangue , Ligante RANK/sangue , Absorciometria de Fóton , Adulto , Idoso , Fosfatase Alcalina/sangue , Densidade Óssea/fisiologia , Feminino , Humanos , Pessoa de Meia-Idade , Osteoporose/enzimologia , Estatísticas não Paramétricas , Adulto JovemRESUMO
OBJECTIVE: To determine the value of pancreatic stone protein in predicting sepsis-related postoperative complications and death in the ICU. DESIGN: A prospective cohort study of postoperative patients admitted to the ICU. Blood samples for analysis were taken within 3 hours from admission to the ICU including pancreatic stone protein, white blood cell counts, C-reactive protein, interleukin-6, and procalcitonin. The Mannheim Peritonitis Index and Acute Physiology and Chronic Health Evaluation II clinical scores were also determined. Univariate and multivariate analyses were performed to determine the diagnostic accuracy and independent predictors of death in the ICU [Clinicaltrials.gov, NCT01465711]. SETTING: An adult medical-surgical ICU in a teaching hospital in Germany. PATIENTS: Ninety-one consecutive postoperative patients with proven diagnosis of secondary peritonitis admitted to the ICU were included in the study from August 17, 2007, to February 8, 2010. INTERVENTIONS: Peripheral vein blood sampling. MEASUREMENTS AND MAIN RESULTS: Univariate analysis demonstrated that pancreatic stone protein has the highest diagnostic accuracy for complications and is the best predictor for death in the ICU. Pancreatic stone protein had the highest overall efficacy in predicting death with an odds ratio of 4.0 vs. procalcitonin (odds ratio 3.2), interleukin-6 (odds ratio 2.8), C-reactive protein (odds ratio 1.3), and WBCs (odds ratio 1.4). By multivariate analysis, pancreatic stone protein was the only independent predictor of death. CONCLUSIONS: In a population of patients with sepsis-related complications, serum-pancreatic stone protein levels demonstrate a high diagnostic accuracy to discriminate the severity of peritonitis and to predict death in the ICU. This test could be of value in the clinical diagnosis and therapeutic decision making in the ICU.
Assuntos
Estado Terminal/mortalidade , Unidades de Terapia Intensiva , Litostatina/sangue , Peritonite/diagnóstico , Peritonite/mortalidade , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina , Estudos de Coortes , Feminino , Alemanha , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Peritonite/sangue , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Precursores de Proteínas/sangue , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Análise de SobrevidaRESUMO
Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.
Assuntos
Lesão Pulmonar Aguda/etiologia , Interleucina-6/fisiologia , Pancreatite/complicações , Células Acinares/metabolismo , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Ácidos Aminossalicílicos/farmacologia , Animais , Benzenossulfonatos/farmacologia , Quimiocina CXCL1/metabolismo , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Pâncreas/patologia , Pancreatite/imunologia , Pancreatite/metabolismo , Pancreatite/patologia , Compostos de Fenilureia/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases. DESIGN: Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 µg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies. RESULTS: Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini. CONCLUSIONS: The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings from the present work further suggest that targeting TNFα, for which pharmaceutical agents are readily available, could be an effective treatment strategy that directly addresses the cellular causes of pancreatitis.
Assuntos
Células Acinares/patologia , Caspase 3/metabolismo , Catepsina B/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Western Blotting , Antígenos CD18/imunologia , Movimento Celular , Ceruletídeo/efeitos adversos , Ativação Enzimática , Leucócitos/fisiologia , Camundongos , Necrose/patologia , Pancreatite Necrosante Aguda/induzido quimicamente , Pancreatite Necrosante Aguda/enzimologia , Pancreatite Necrosante Aguda/patologia , Peptídeo Hidrolases/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: The transcription factor nuclear factor-κB (NF-κB) (a heterodimer of NF-κB1p50 and RelA) is activated rapidly in acute pancreatitis (AP). However, it is not clear whether NF-κB promotes or protects against AP. We used the NF-κB inhibitor protein, inhibitor of κB (IκB)α, to study the roles of NF-κB in the development of AP in mice. METHODS: IκBα or the combination of IκBα and RelA selectively were deleted from pancreas of mice using the Cre/locus of cross-over P strategy; cerulein or L-arginine were used to induce AP. We performed microarray analyses of the IκBα- and RelA-deficient pancreata. DNA from healthy individuals and patients with acute or chronic pancreatitis were analyzed for variants in coding regions of alpha-1-antichymotrypsin. RESULTS: Mice with pancreas-specific deletion of IκBα had constitutive activation of RelA and a gene expression profile consistent with NF-κB activation; development of AP in these mice was attenuated and trypsin activation was impaired. However, AP was fully induced in mice with pancreas-specific deletion of IκBα and RelA. By using genome-wide expression analysis, we identified a cluster of NF-κB-regulated genes that might protect against the development of AP. The serine protease inhibitor 2A (Spi2a) was highly up-regulated in IκBα-deficient mice. Lentiviral-mediated expression of Spi2A reduced the development of AP in C57BL/6 and RelA-deficient mice. However, we did not correlate any variants of alpha-1-antichymotrypsin, the human homologue of Spi2a, with acute or chronic pancreatitis. CONCLUSIONS: Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and reduces AP induction and trypsin activation in mice after administration of cerulein or L-arginine. Constitutive activation of RelA up-regulates Spi2A, which protects mice against the development of AP.
Assuntos
Proteínas I-kappa B/genética , NF-kappa B/metabolismo , Pancreatite/genética , Pancreatite/metabolismo , Serpinas/genética , Fator de Transcrição RelA/genética , alfa 1-Antiquimotripsina/genética , Células Acinares , Animais , Arginina , Ceruletídeo , Citosol/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Vetores Genéticos , Genótipo , Proteínas I-kappa B/metabolismo , Lentivirus , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Inibidor de NF-kappaB alfa , Proteínas Nucleares/metabolismo , Pâncreas/enzimologia , Pancreatite/induzido quimicamente , Pancreatite/patologia , Fosforilação , Serpinas/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Tripsina/metabolismo , Regulação para CimaRESUMO
OBJECTIVES: Cathepsin K (CatK) is expressed in high levels in osteoplasts and therefore plays an important role in bone resorption. Thus CatK serum levels may be useful in the diagnosis of chronic bone disorders such as osteopenia and osteoporosis. Therefore we aimed at studying CatK levels in women putatively free of known skeletal disorders. STUDY DESIGN: In total, 121 voluntary women, 27 premenopausal women aged between 20 and 45 years, and 94 postmenopausal women aged 59-81 years, all free of known skeletal disorders were included. All women underwent bone density measurement, routine labor parameter and measurement of serum CatK levels. MAIN OUTCOME MEASURES: Based on WHO criteria, women were stratified in four groups (premenopausal: healthy; postmenopausal: healthy, osteopenia, osteoporosis), and their CatK levels were statistically analyzed. RESULTS: Using WHO criteria 21 postmenopausal women had normal bone mineral density (BMD), 49 had osteopenia and 24 had osteoporosis. All 27 premenopausal women had normal BMD. There were no significant differences in CatK between these groups. ROC analysis resulted in poor diagnostic validity of CatK, where the area under curve was 0.544. There was no correlation neither between CatK and other biomarkers as C-telopeptide crosslaps (CTX) or bone-specific alkaline phosphatase (BAP) nor between CatK and age. CONCLUSIONS: Serum levels of CatK are not suitable to differentiate women with osteoporosis from healthy subjects.
Assuntos
Densidade Óssea , Doenças Ósseas Metabólicas/diagnóstico , Reabsorção Óssea/sangue , Catepsina K/sangue , Osteoporose Pós-Menopausa/diagnóstico , Pós-Menopausa , Pré-Menopausa , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/sangue , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/epidemiologia , Feminino , Humanos , Incidência , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/epidemiologia , Curva ROC , Valores de Referência , Adulto JovemRESUMO
OBJECTIVES: Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP. METHODS: To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis. RESULTS: Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen. CONCLUSIONS: These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.