RESUMEN
OBJECTIVES: Lymphangioleiomyomatosis (LAM) patients with severe lung disease may be considered for lung transplantation. Clinical, physiologic, and quality of life data are usually employed for referral. The aim of this study was to determine whether computed tomographic measurement of lung volume occupied by cysts (cyst score) complemented clinical and physiologic data in supporting referral for transplantation. METHODS: Forty-one patients were studied. Pre-referral clinical data, pulmonary function tests, exercise testing, and high-resolution computed tomography (HRCT) scans were obtained. From HRCT, a computer-aided diagnostic program was employed to calculate cyst scores. These data were compared to those of 41 age-matched LAM patients not referred for lung transplantation. RESULTS: Cyst score, and % predicted FEV1 and DLCO were respectively, 48.1 ± 9.4%, 36.5 ± 9.1%, and 35.0 ± 10.7%. For the control group, cyst score, FEV1, and DLCO were respectively, 14.8 ± 8.3%, 77.2 ± 20.3%, and 66.7 ± 19.3%. Cyst score values showed a normal distribution. However, the frequency distribution of FEV1 was skewed to the right while the distribution of DLCO was bimodal. Correlations between cyst score and FEV1 and DLCO for the study group were respectively, r = - 0.319 and r = - 0.421. CONCLUSIONS: LAM patients referred for lung transplantation had nearly 50% of lungs occupied by cysts. Correlations between cyst score and FEV1 or DLCO were weak; as shown previously, DLCO was better related to cyst number while FEV1 had a better association with cyst size. Given its normal distribution, cyst score measurements may assist in evaluation of pre-transplant severity of lung disease before referral for transplantation.
Asunto(s)
Quistes , Enfermedades Pulmonares , Neoplasias Pulmonares , Linfangioleiomiomatosis , Computadores , Quistes/diagnóstico por imagen , Humanos , Pulmón/diagnóstico por imagen , Neoplasias Pulmonares/complicaciones , Linfangioleiomiomatosis/complicaciones , Linfangioleiomiomatosis/diagnóstico por imagen , Calidad de Vida , Derivación y Consulta , Índice de Severidad de la EnfermedadRESUMEN
Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease with mortality driven primarily by respiratory failure. Patients with LAM frequently have respiratory infections, suggestive of a dysregulated microbiome. Here we demonstrate that end-stage LAM patients have a distinct microbiome signature compared to patients with end-stage chronic obstructive pulmonary disease.
Asunto(s)
Pulmón/microbiología , Linfangioleiomiomatosis/microbiología , Microbiota , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Infecciones del Sistema Respiratorio/microbiología , Progresión de la Enfermedad , Disbiosis , Humanos , Enfermedades Pulmonares Fúngicas/diagnóstico , Enfermedades Pulmonares Fúngicas/microbiología , Linfangioleiomiomatosis/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Infecciones del Sistema Respiratorio/diagnóstico , RibotipificaciónRESUMEN
Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2+/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/metabolismo , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Broncodilatadores/farmacología , Dominio Catalítico , Proliferación Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Progresión de la Enfermedad , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Isoproterenol/farmacología , Persona de Mediana Edad , Análisis Multivariante , Fosforilación , Pruebas de Función Respiratoria , Estudios Retrospectivos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Piel/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, affecting lungs, kidneys, and lymphatics. It is caused by the proliferation of abnormal smooth muscle-like LAM cells, with mutations and loss of heterozygosity in the TSC1 or, more frequently, TSC2 genes. Isolated pulmonary LAM cells have been difficult to maintain in culture, and most studies of LAM lung cells involve mixtures of TSC2 wild-type and TSC2-null cells. A clonal population of LAM lung cells has not been established, making analysis of the cells challenging. Cell lines have been established from angiomyolipomas, a common manifestation of LAM, and from tumors from patients with TSC. Circulating LAM cells have also been isolated from blood and other body fluids. LAM cells may also be identified in clusters apparently derived from lymphatic vessels. Genetics, patterns of antigen expression, and signaling pathways have been studied in LAM lung tissue and in LAM cell models, although rarely all in the same study. We show here that LAM cells manifest differences in these characteristics, depending on the source investigated, suggesting further studies.
Asunto(s)
Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Linfangioleiomiomatosis/genética , Mutación , Piel/patología , Células Tumorales CultivadasRESUMEN
Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (â¼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor-binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors.
Asunto(s)
Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Fibrosis Pulmonar Idiopática/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Fibroblastos/citología , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Inmunoprecipitación , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor-related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors.
Asunto(s)
Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/patología , Osteoprotegerina/metabolismo , Proteínas Supresoras de Tumor/genética , Adulto , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiocina CCL2/sangre , Citometría de Flujo , Voluntarios Sanos , Humanos , Pérdida de Heterocigocidad , Pulmón/metabolismo , Pulmón/patología , Linfangioleiomiomatosis/sangre , Linfangioleiomiomatosis/metabolismo , Microdisección , Persona de Mediana Edad , Metástasis de la Neoplasia , Osteoprotegerina/sangre , Osteoprotegerina/genética , Receptores de Superficie Celular/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismo , Antígeno gp100 del Melanoma/metabolismoRESUMEN
ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-ß receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Western Blotting , Células Cultivadas , Humanos , Hidrólisis , Ratones , Ratones Noqueados , Microscopía Fluorescente , ARN Mensajero/genética , Receptores de Factores de Crecimiento/genéticaRESUMEN
Lymphangioleiomyomatosis (LAM) is a multisystem disease affecting primarily women, characterised in the lung by proliferation of LAM cells, abnormal smooth muscle-like cells with dysfunctional tuberous sclerosis complex genes. This dysfunction results in activation of mechanistic target of rapamycin (mTOR), leading to LAM cell proliferation. Sirolimus (rapamycin) is the only United States Food and Drug Administration-approved treatment for pulmonary LAM, resulting in decreased LAM cell growth/size and stabilised lung function [1].
Asunto(s)
GTP Fosfohidrolasas/genética , Histiocitosis de Células de Langerhans , Proteínas de la Membrana/genética , Células Neoplásicas Circulantes , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Antígenos CD/metabolismo , Antígenos CD1 , Biomarcadores/análisis , Femenino , GTP Fosfohidrolasas/análisis , Histiocitosis de Células de Langerhans/sangre , Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/genética , Humanos , Células de Langerhans/inmunología , Lectinas Tipo C/metabolismo , Pulmón/diagnóstico por imagen , Masculino , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas B-raf/análisis , Tomografía Computarizada por Rayos X/métodosRESUMEN
ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/aislamiento & purificación , Citosol/efectos de los fármacos , Citosol/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/aislamiento & purificación , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Inhibidores de Fosfodiesterasa 3 , Unión Proteica , ARN Interferente Pequeño/genéticaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating respiratory disease whose pathogenesis is poorly understood. In IPF, the lung parenchyma undergoes extensive remodeling. We hypothesized that lymphangiogenesis is part of lung remodeling and sought to characterize pathways leading to lymphangiogenesis in IPF. We found that the diameter of lymphatic vessels in alveolar spaces in IPF lung tissue correlated with disease severity, suggesting that the alveolar microenvironment plays a role in the lymphangiogenic process. In bronchoalveolar lavage fluid (BALF) from subjects with IPF, we found short-fragment hyaluronic acid, which induced migration and proliferation of lymphatic endothelial cells (LECs), processes required for lymphatic vessel formation. To determine the origin of LECs in IPF, we isolated macrophages from the alveolar spaces; CD11b(+) macrophages from subjects with IPF, but not those from healthy volunteers, formed lymphatic-like vessels in vitro. Our findings demonstrate that in the alveolar microenvironment of IPF, soluble factors such as short-fragment hyaluronic acid and cells such as CD11b(+) macrophages contribute to lymphangiogenesis. These results improve our understanding of lymphangiogenesis and tissue remodeling in IPF and perhaps other fibrotic diseases as well.
Asunto(s)
Fibrosis Pulmonar Idiopática/complicaciones , Fibrosis Pulmonar Idiopática/patología , Linfangiogénesis , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Antígeno CD11b/metabolismo , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Salud , Humanos , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/farmacología , Linfangiogénesis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Neovascularización Fisiológica/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: Lymphangioleiomyomatosis (LAM) and pulmonary Langerhans cell histiocytosis (PLCH) are cystic lung diseases in which a neoplastic cell is thought to be responsible for disease pathogenesis. The neoplastic LAM cell has mutations in the TSC genes, TSC1 or TSC2, whereas the neoplastic PLCH cell may have mutations in several genes (eg, BRAF, NRAS, MAP2K1). These mutations are not specific for PLCH and have been described in multiple cancers. TSC1 or TSC2 mutations and loss of heterozygosity (LOH) have also been described in cancers. RESEARCH QUESTION: Is TSC2 LOH specific to LAM or is it also found in PLCH? STUDY DESIGN AND METHODS: We recruited patients with LAM (n = 53) and healthy volunteers (n = 22) and compared the presence of cells with TSC2 LOH with patients with PLCH (n = 12). Blood and urine samples were collected for analysis. Fluorescence-activated cell sorting (FACS) was used to identify subpopulations of cells from blood and urine samples. We isolated CD45-CD235a-, CD45-CD235a+, and CD45+CD235a- cells from blood after density gradient separation. Cells were screened for TSC2 LOH at five microsatellites markers (ie, kg8, D16S3395, D16S3024, D16S521, D16S291). We obtained four cell subpopulations from urine (ie, CD44v6+CD9+, CD44v6+CD9-, CD44v6-CD9+, CD44v6-CD9-). RESULTS: Using FACS, cells were isolated from blood and urine from patients with PLCH that showed TSC2 LOH. Healthy volunteers did not have cells with TSC2 LOH. As a control, cells isolated from blood and urine from patients with LAM gave results similar to those reported previously. These data show that TSC2 LOH is found in patients with cystic lung diseases with potential neoplastic characteristics, and in patients with cancer. INTERPRETATION: The presence of TSC2 LOH in circulating cells is not specific for LAM. The data suggest that chromosomal abnormalities affecting the TSC2 gene are found in other diseases associated with cells having cancer-like neoplastic cells.
Asunto(s)
Histiocitosis de Células de Langerhans , Enfermedades Pulmonares , Linfangioleiomiomatosis , Histiocitosis de Células de Langerhans/genética , Humanos , Pérdida de Heterocigocidad , Enfermedades Pulmonares/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2) and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). Here, we report that E26 transformation-specific (ETS) variant transcription factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. ETV2 nuclear localization in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP) mRNA and protein expression, partially reversing the observed down-regulation of PARPBP expression induced by mTORC1 blockade during treatment with both Syk and mTORC1 inhibitors. In addition, silencing Etv2 or Parpbp in Tsc2-deficient cells induced ER stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2, lending support to the translational relevance of our findings. In conclusion, we report a novel ETV2 signaling axis unique to Syk inhibition that promotes a cytocidal response in Tsc2-deficient cells and therefore maybe a potential alternative therapeutic target in LAM.
Asunto(s)
Linfangioleiomiomatosis , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas de Unión al ADN/genética , Estrés del Retículo Endoplásmico , Humanos , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Factores de Transcripción/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2-/-, but not of TSC2+/-, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells.
Asunto(s)
División Celular/fisiología , Eritropoyetina/farmacología , Eritropoyetina/fisiología , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Mutación , Proteínas Supresoras de Tumor/genética , División Celular/efectos de los fármacos , Progresión de la Enfermedad , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/metabolismo , Linfangioleiomiomatosis/patología , Transducción de Señal/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/efectos de los fármacosRESUMEN
Patients with tuberous sclerosis complex (TSC) develop hamartomatous tumors showing loss of function of the tumor suppressor TSC1 (hamartin) or TSC2 (tuberin) and increased angiogenesis, fibrosis, and abundant mononuclear phagocytes. To identify soluble factors with potential roles in TSC tumorigenesis, we screened TSC skin tumor-derived cells for altered gene and protein expression. Fibroblast-like cells from 10 angiofibromas and five periungual fibromas produced higher levels of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein than did fibroblasts from the same patient's normal skin. Conditioned medium from angiofibroma cells stimulated chemotaxis of a human monocytic cell line to a greater extent than conditioned medium from TSC fibroblasts, an effect blocked by neutralizing MCP-1-specific antibody. Overexpression of MCP-1 seems to be caused by loss of tuberin function because Eker rat embryonic fibroblasts null for Tsc2 (EEF Tsc2(-/-)) produced 28 times as much MCP-1 protein as did EEF Tsc2(+/+) cells; transient expression of WT but not mutant human TSC2 by EEF Tsc2(-/-) cells inhibited MCP-1 production; and pharmacological inhibition of the Rheb-mTOR pathway, which is hyperactivated after loss of TSC2, decreased MCP-1 production by EEF Tsc2(-/-) cells. Together these findings suggest that MCP-1 is an important paracrine factor for TSC tumorigenesis and may be a new therapeutic target.
Asunto(s)
Quimiocina CCL2/metabolismo , Fibroma/metabolismo , Regulación Neoplásica de la Expresión Génica , Comunicación Paracrina/efectos de los fármacos , Esclerosis Tuberosa/metabolismo , Análisis de Varianza , Animales , Anticuerpos/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Fibroma/etiología , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Esclerosis Tuberosa/complicaciones , Proteína 2 del Complejo de la Esclerosis Tuberosa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM), a rare cystic lung disease with multi-organ involvement, occurs primarily in women of childbearing age. LAM can present sporadically or in association with tuberous sclerosis complex (TSC). Loss of lung function in patients with LAM can be attributed to the dysregulated growth of LAM cells, with dysfunctional TSC1 or TSC2 genes, which encode hamartin and tuberin, respectively, leading to hyperactivation of the mammalian target of rapamycin (mTOR). LAM cells are smooth muscle-like cells that express melanoma antigens such as gp100, a splice variant of the Pmel17 gene. Tuberin and hamartin form heterodimers that act as negative regulators of mTOR. Lack of TSC2 function, as occurs in LAM cells, leads to the production of the chemokine CCL2/monocyte chemotactic protein 1 (MCP-1), which increases LAM cell mobility. Although many chemokines and their receptors could influence LAM cell mobilization, we propose that a positive-feedback loop is generated when dysfunctional TSC2 is present in LAM cells. We identified a group of chemokine receptors that is expressed in LAM cells and differs from those on smooth muscle and melanoma cells (Malme-3M). Chemokines have been implicated in tumor metastasis, and our data suggest a role for chemokines in LAM cell mobilization and thereby in the pathogenesis of LAM.
Asunto(s)
Movimiento Celular , Quimiocinas/inmunología , Linfangioleiomiomatosis/inmunología , Animales , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/patología , Metástasis de la Neoplasia/inmunología , Receptores de Quimiocina/inmunologíaRESUMEN
Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.