RESUMEN
The E3 ubiquitin ligase thyroid hormone receptor interacting protein 12 (TRIP12) has been implicated in pancreatic adenocarcinoma (PDAC) through its role in mediating the degradation of pancreas transcription factor 1a (PTF1a). PTF1a is a transcription factor essential for the acinar differentiation state that is notably diminished during the early steps of pancreatic carcinogenesis. Despite these findings, the direct involvement of TRIP12 in the onset of pancreatic cancer has yet to be established. In this study, we demonstrated that TRIP12 protein was significantly upregulated in human pancreatic preneoplastic lesions. Furthermore, we observed that TRIP12 overexpression varied within PDAC samples and PDAC-derived cell lines. We further demonstrated that TRIP12 was required for PDAC-derived cell growth and for the expression of E2F-targeted genes. Acinar-to-ductal cell metaplasia (ADM) is a reversible process that reflects the high plasticity of acinar cells. ADM becomes irreversible in the presence of oncogenic Kras mutations and leads to the formation of preneoplastic lesions. Using two genetically modified mouse models, we showed that a loss of TRIP12 prevented acini from developing ADM in response to pancreatic injury. With two additional mouse models, we further discovered that a depletion of TRIP12 prevented the formation of KrasG12D-induced preneoplastic lesions and impaired metastasis formation in the presence of mutated KrasG12D and Trp53R172H genes. In summary our study identified an overexpression of TRIP12 from the early stages of pancreatic carcinogenesis and proposed this E3 ubiquitin ligase as a novel regulator of acinar plasticity with an important dual role in initiation and metastatic steps of PDAC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Células Acinares , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ubiquitina-Proteína Ligasas , Animales , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/enzimología , Humanos , Células Acinares/patología , Células Acinares/metabolismo , Células Acinares/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/enzimología , Metaplasia/patología , Metaplasia/metabolismo , Plasticidad de la Célula , Carcinogénesis/genética , Carcinogénesis/metabolismo , Ratones , Línea Celular Tumoral , Proliferación Celular , Ratones Noqueados , Regulación Neoplásica de la Expresión Génica , Lesiones Precancerosas/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/enzimología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Transformación Celular Neoplásica/metabolismo , Proteínas PortadorasRESUMEN
Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Adenocarcinoma , Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Fibroblastos Asociados al Cáncer/patología , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. METHODS: Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. RESULTS: Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. CONCLUSIONS: We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.
Asunto(s)
Fibroblastos Asociados al Cáncer/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Secretoma/efectos de los fármacos , Somatostatina/análogos & derivados , Anciano , Anciano de 80 o más Años , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/secundario , Femenino , Hormonas/farmacología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Somatostatina/farmacologíaRESUMEN
Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid ß-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.
Asunto(s)
Autofagia/fisiología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia/metabolismo , Mitocondrias/metabolismo , Animales , Autofagia/genética , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Citometría de Flujo , Humanos , Leucemia/genética , Leucemia Mieloide Aguda/patología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Lipogénesis/genética , Lipogénesis/fisiología , Ratones , Mitocondrias/genética , Oxidación-Reducción , Fosforilación OxidativaRESUMEN
BACKGROUND & AIMS: The KRAS gene is mutated in most pancreatic ductal adenocarcinomas (PDAC). Expression of this KRAS oncoprotein in mice is sufficient to initiate carcinogenesis but not progression to cancer. Activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is required for KRAS for induction and maintenance of PDAC in mice. The somatostatin receptor subtype 2 (sst2) inhibits PI3K, but sst2 expression is lost during the development of human PDAC. We investigated the effects of sst2 loss during KRAS-induced PDAC development in mice. METHODS: We analyzed tumor growth in mice that expressed the oncogenic form of KRAS (KRAS(G12D)) in pancreatic precursor cells, as well as sst2+/- and sst2-/-, and in crossed KRAS(G12D);sst2+/- and KRAS(G12D);sst2-/- mice. Pancreatic tissues and acini were collected and assessed by histologic, immunoblot, immunohistochemical, and reverse-transcription polymerase chain reaction analyses. We also compared protein levels in paraffin-embedded PDAC samples from patients vs heathy pancreatic tissues from individuals without pancreatic cancer. RESULTS: In sst2+/- mice, PI3K was activated and signaled via AKT (PKB; protein kinase B); when these mice were crossed with KRAS(G12D) mice, premalignant lesions, tumors, and lymph node metastases developed more rapidly than in KRAS(G12D) mice. In crossed KRAS(G12D);sst2+/- mice, activation of PI3K signaling via AKT resulted in activation of nuclear factor-κB (NF-κB), which increased KRAS activity and its downstream pathways, promoting initiation and progression of neoplastic lesions. We found this activation loop to be mediated by PI3K-induced production of the chemokine CXCL16. Administration of a CXCL16-neutralizing antibody to KRAS(G12D) mice reduced activation of PI3K signaling to AKT and NF-κB, blocking carcinogenesis. Levels of CXCL16 and its receptor CXCR6 were significantly higher in PDAC tissues and surrounding acini than in healthy pancreatic tissues from mice or human beings. In addition, expression of sst2 was progressively lost, involving increased PI3K activity, in mouse lesions that expressed KRAS(G12D) and progressed to PDAC. CONCLUSIONS: Based on analyses of mice, loss of sst2 from pancreatic tissues activates PI3K signaling via AKT, leading to activation of NF-κB, amplification of oncogenic KRAS signaling, increased expression of CXCL16, and pancreatic tumor formation. CXCL16 might be a therapeutic target for PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Proliferación Celular , Quimiocina CXCL6/metabolismo , Mutación , Neoplasias Pancreáticas/enzimología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Somatostatina/deficiencia , Transducción de Señal , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/secundario , Estudios de Casos y Controles , Línea Celular Tumoral , Quimiocina CXCL16 , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Metástasis Linfática , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Depuradores/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral , Regulación hacia ArribaRESUMEN
It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial-mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and alpha-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues.
Asunto(s)
Células Epiteliales/citología , Mesodermo/citología , Páncreas Exocrino/citología , Células Madre/citología , Adulto , Amilasas/biosíntesis , Biomarcadores/metabolismo , Desdiferenciación Celular , Proliferación Celular , Transdiferenciación Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Células Epiteliales/metabolismo , Glucagón/biosíntesis , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Mesodermo/metabolismo , Persona de Mediana Edad , Páncreas Exocrino/metabolismo , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Células Madre/metabolismo , Adulto JovenRESUMEN
The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.
Asunto(s)
Anhidrasa Carbónica IV/metabolismo , Conductos Pancreáticos/metabolismo , Anhidrasa Carbónica IV/biosíntesis , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente , Glicosilfosfatidilinositoles/metabolismo , Aparato de Golgi/metabolismo , Humanos , Microscopía Inmunoelectrónica , Conductos Pancreáticos/citología , Vesículas Secretoras/metabolismoRESUMEN
Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.
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Fosfatidilinositol 3-Quinasas/fisiología , Receptores de Somatostatina/fisiología , Somatostatina/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular , Activación Enzimática , Femenino , Humanos , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Unión Proteica , Receptores de Somatostatina/genética , Transducción de Señal , Resonancia por Plasmón de Superficie , Trasplante Heterólogo , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Fibrosis Quística/metabolismo , Conductos Pancreáticos/metabolismo , Anhidrasa Carbónica IV/metabolismo , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Conductos Pancreáticos/ultraestructura , Transporte de Proteínas , TransfecciónRESUMEN
Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression is enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.
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Conexina 43/biosíntesis , Conexinas/biosíntesis , Uniones Comunicantes/fisiología , Neoplasias/metabolismo , Neoplasias/patología , Ribosomas/metabolismo , Regiones no Traducidas 5'/genética , Comunicación Celular , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Humanos , Neoplasias/genética , Biosíntesis de Proteínas/efectos de los fármacos , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sirolimus/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Alveolar type II pneumocytes are thought to be progenitor cells capable of self-renewal and differentiation into type I pneumocytes. Nevertheless, the existence of an alveolar stem cell has been postulated. In lungs from patients with cystic fibrosis, the alveolar epithelium is damaged with ulceration and subsequent regeneration. We characterized alveolar modifications histologically and immunohistochemically in the pulmonary tissue of a patient homozygous for the DeltaF508 mutation. Alveoli were of variable size and surrounded by an inflammatory infiltrate. They were lined by a continuous layer of cuboidal cells with very weak proliferative activity. These cells resembled type II pneumocytes. They expressed thyroid transcription factor-1, cystic fibrosis transmembrane conductance regulator, cytokeratin 7 and contained lamellar bodies. Weak expression of cytokeratin 5 considered to be a marker of progenitor cells of the bronchial and bronchiolar epithelium was detected. Explantation of this alveolar epithelium produced primary cultures and subcultures of epithelial cells that had acquired proliferative properties showing signs of dedifferentiation with a loss of lamellar bodies and a lack of expression of thyroid transcription factor-1. Persistence of the expression of cytokeratin 7 and a strong expression of cytokeratin 5 were observed. The culture conditions were thought to have circumvented the inhibition of proliferation observed in vivo due to the inflammatory peri-alveolar environment. They thus favored the multiplication of a population of cells co-expressing cytokeratin 5 and certain characteristics of type II pneumocytes. The presence of these cells of intermediate phenotype is indicative of the existence of immature precursors for type II pneumocytes.
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Fibrosis Quística/metabolismo , Pulmón/citología , Alveolos Pulmonares , Células Madre/metabolismo , Biomarcadores , Células Cultivadas , Niño , Fibrosis Quística/genética , Fibrosis Quística/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Queratina-7 , Queratinas/metabolismo , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/ultraestructura , Células Madre/citologíaRESUMEN
The high concentration of HCO(3)(-) ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl(-)/HCO(3)(-) exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors ( n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH(2) antibody directed against the NH(2) terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.
Asunto(s)
Anhidrasa Carbónica IV/metabolismo , Conductos Pancreáticos/enzimología , Bicarbonatos/metabolismo , Anhidrasa Carbónica IV/química , Membrana Celular/enzimología , Separación Celular , Células Cultivadas , Humanos , Técnicas para Inmunoenzimas , Conductos Pancreáticos/citología , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.