RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a devastating metastatic disease for which better therapies are urgently needed. Macrophages enhance metastasis in many cancer types; however, the role of macrophages in PDAC liver metastasis remains poorly understood. Here we found that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting inflammatory monocytes to the liver. Mechanistically, we demonstrate that granulin secretion by metastasis-associated macrophages (MAMs) activates resident hepatic stellate cells (hStCs) into myofibroblasts that secrete periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment or genetic depletion of granulin reduced hStC activation and liver metastasis. Interestingly, we found that circulating monocytes and hepatic MAMs in PDAC patients express high levels of granulin. These findings suggest that recruitment of granulin-expressing inflammatory monocytes plays a key role in PDAC metastasis and may serve as a potential therapeutic target for PDAC liver metastasis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cirrosis Hepática/patología , Neoplasias Hepáticas/secundario , Macrófagos/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Células Estrelladas Hepáticas/patología , Humanos , Inflamación/patología , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Macrófagos/metabolismo , Ratones , Monocitos/metabolismo , Monocitos/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Metástasis de la Neoplasia , Progranulinas , Neoplasias PancreáticasRESUMEN
blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.
Asunto(s)
Anomalías Múltiples/genética , Catarata/congénito , Catarata/genética , Córnea/anomalías , Hipogonadismo/genética , Infertilidad Masculina/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Mutación , Atrofia Óptica/genética , Proteínas de Unión al GTP rab1/genética , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/patología , Catarata/diagnóstico , Catarata/metabolismo , Línea Celular , Córnea/metabolismo , Análisis Mutacional de ADN , Facies , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/metabolismo , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/metabolismo , Cristalino/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Microcefalia/diagnóstico , Microcefalia/metabolismo , Atrofia Óptica/diagnóstico , Atrofia Óptica/metabolismo , Linaje , Fenotipo , Alineación de Secuencia , Testículo/patología , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Síndrome de Hermanski-Pudlak/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Factores de Intercambio de Guanina Nucleótido/genética , Síndrome de Hermanski-Pudlak/genética , Humanos , Melanosomas , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Proteínas/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions.
Asunto(s)
Proteínas ADAM/metabolismo , Uniones Adherentes/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cadherinas/metabolismo , Movimiento Celular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína ADAM10 , Transporte Biológico , Endosomas/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Transporte de Proteínas/fisiología , Transferrina/metabolismo , Proteínas de Unión al GTP rab4/metabolismoRESUMEN
A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Proteínas de Unión al GTP rab/química , Transporte Biológico , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/fisiología , Células HeLa , Humanos , Lisosomas/metabolismo , Estructura Terciaria de Proteína , Toxina Shiga/metabolismo , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/fisiología , Red trans-Golgi/metabolismoRESUMEN
Chemokine-induced activation of alpha4beta1 and alphaLbeta2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of alphaLbeta2; engagement of alpha4beta1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in alphaLbeta2 activation and determine how it is corrected. We show here that the alphaLbeta2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both alpha4beta1 and alphaLbeta2 is defective, autocrine VEGF and chemokine are necessary to activate alpha4beta1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for alphaLbeta2 activation for motility and TEM. The present study not only clarifies the nature of the alphaLbeta2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL.