RESUMO
Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.
Assuntos
Neoplasias Pulmonares , Linfangioleiomiomatose , Feminino , Humanos , Células Epiteliais Alveolares/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/metabolismo , Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Mastócitos/metabolismo , Triptases/metabolismo , Adulto , Animais , Biomarcadores Tumorais/genética , Quimiocinas/metabolismo , Progressão da Doença , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/patologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1 ), higher diffusion in the lung for carbon monoxide (DLCO ) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI -1.11 to -0.18) and a 0.50% decrease in DLCO (95% CI -0.96 to -0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho-ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.
Assuntos
Progressão da Doença , Pulmão/patologia , Linfangioleiomiomatose , Neoplasias de Células Epitelioides Perivasculares , Adulto , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Catepsina K/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/metabolismo , Linfangioleiomiomatose/patologia , Pessoa de Meia-Idade , Neoplasias de Células Epitelioides Perivasculares/metabolismo , Neoplasias de Células Epitelioides Perivasculares/patologia , Estudos Prospectivos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of extracellular matrix (ECM) proteins and fibroblast proliferation. ECM cross-linking enzymes have been implicated in fibrotic diseases, and we hypothesized that the ECM in IPF is abnormally cross-linked, which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross-linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase-like 1 (LOXL1), LOXL2, LOXL3, and LOXL4 were expressed equally in control and IPF-derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. LOXL2-, transglutaminase 2-, and transglutaminase-generated cross-links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity than those grown on control ECM. IPF-derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminase activity during ECM formation affected ECM structure as visualized by electron microscopy, and it reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not of lysyl oxidase, activity enhanced the turnover of ECM in vitro. In bleomycin-treated mice, during the postinflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole-lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross-linking, which contributes to increased fibroblast growth and resistance to normal ECM turnover to drive lung fibrosis.
Assuntos
Remodelação das Vias Aéreas , Proliferação de Células , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Aminoácido Oxirredutases/metabolismo , Animais , Bleomicina , Adesão Celular , Células Cultivadas , Cistamina/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/prevenção & controle , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase , Proteólise , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismoRESUMO
Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM.
Assuntos
Catepsina K/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Linfangioleiomiomatose/metabolismo , Peptídeo Hidrolases/metabolismo , Adulto , Comunicação Celular , Linhagem Celular , Técnicas de Cocultura , Feminino , Fibroblastos/patologia , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/patologiaRESUMO
RATIONALE: Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of people with asthma. OBJECTIVES: To investigate whether MMP-1 could be activated by mast cells and increase asthma severity. METHODS: Patients with stable asthma and healthy control subjects underwent spirometry, methacholine challenge, and bronchoscopy, and their airway smooth muscle cells were grown in culture. A second asthma group and control subjects had symptom scores, spirometry, and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extracellular matrix was prepared from decellularized airway smooth muscle cultures. MMP-1 protein and activity were assessed. MEASUREMENTS AND MAIN RESULTS: Airway smooth muscle cells generated pro-MMP-1, which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extracellular matrix, which enhanced subsequent airway smooth muscle growth by 1.5-fold (P < 0.05), which was dependent on MMP-1 activation. In asthma, airway pro-MMP-1 was 5.4-fold higher than control subjects (P = 0.002). Mast cell numbers were associated with airway smooth muscle proliferation and MMP-1 protein associated with bronchial hyperresponsiveness. During exacerbations, MMP-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. CONCLUSIONS: MMP-1 is activated by mast cell tryptase resulting in a proproliferative extracellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, MMP-1 levels are associated with bronchial hyperresponsiveness, and MMP-1 activation are associated with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing MMP activation, airway smooth muscle growth, and airway responsiveness.
Assuntos
Asma/metabolismo , Asma/fisiopatologia , Brônquios/metabolismo , Brônquios/fisiopatologia , Metaloproteinase 1 da Matriz/metabolismo , Músculo Liso/metabolismo , Adulto , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Técnicas de Cultura de Células , Feminino , Humanos , Masculino , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Índice de Gravidade de Doença , EspirometriaRESUMO
BACKGROUND: The Royal College of Physicians and Surgeons of Canada and the College of Family Physicians of Canada mandate that faculty members demonstrate they are evaluating residents on all CanMEDS (Canadian Medical Education Directions for Specialists) roles as part of the accreditation process. Postgraduate Medical Education at the University of Ottawa initiated a 5-year project to develop and implement a comprehensive system to assess the full spectrum of CanMEDS roles. This paper presents the findings from a needs assessment with Program Directors, in order to determine how postgraduate medical faculty can be motivated and supported to evaluate residents on the intrinsic CanMEDS roles. METHODS: Semi-structured individual interviews were conducted with 60 Postgraduate Program Directors in the Faculty of Medicine. Transcribed interviews were analyzed using qualitative analysis. Once the researchers were satisfied the identified themes reflected the views of the participants, the data was assigned to categories to provide rich, detailed, and comprehensive information that would indicate what faculty need in order to effectively evaluate their residents on the intrinsic roles. RESULTS: Findings indicated faculty members need faculty development and shared point of care resources to support them with how to not only evaluate, but also teach, the intrinsic roles. Program Directors expressed the need to collaborate and share resources across departments and national specialty programs. Based on our findings, we designed and delivered workshops with companion eBooks to teach and evaluate residents at the point of care (Developing the Professional, Health Advocate and Scholar). CONCLUSIONS: Identifying stakeholder needs is essential for designing effective faculty development. By sharing resources, faculties can prevent 'reinventing the wheel' and collaborate to meet the Colleges' accreditation requirements more efficiently.
Assuntos
Competência Clínica , Educação de Pós-Graduação em Medicina/normas , Docentes de Medicina/normas , Internato e Residência , Acreditação , Canadá , Avaliação Educacional , Humanos , Pesquisa Qualitativa , Desenvolvimento de PessoalRESUMO
Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.
Assuntos
Pulmão/patologia , Linfangioleiomiomatose/patologia , Células-Tronco Mesenquimais/patologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Imunofluorescência , Humanos , Pulmão/citologia , CamundongosRESUMO
Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the ß1 and ß3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.
Assuntos
Asma/fisiopatologia , Proteínas da Matriz Extracelular/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Asma/genética , Asma/patologia , Bradicinina/farmacologia , Células Cultivadas , Colágeno Tipo I/farmacologia , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos , Immunoblotting , Indóis/farmacologia , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Metaloproteinase 1 da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Interferência de RNA , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tenascina/genética , Tenascina/metabolismo , Tenascina/farmacologia , Vasodilatadores/farmacologiaRESUMO
Lymphangioleiomyomatosis (LAM) is a progressive disease caused by accumulation of metastatic (LAM) cells in the lungs, lymphatics, and the tumor angiomyolipoma (AML). LAM cells have biallelic loss of either tuberous sclerosis complex gene (but predominantly TSC-2) and resultant dysregulation of the mammalian target of rapamycin (mTOR) pathway. Chemokines are associated with neoplastic cell growth, survival, and homing to specific organs and may play similar roles in LAM. Our objective was to study comprehensively the expression and function of chemokine receptors and how their function interacts with dysregulation of the mTOR pathway in LAM and AML. We used RT-PCR and FACS to study receptor expression in primary AML cells and immunohistochemistry to investigate expression in tissues. Chemokine receptor function was analyzed in AML cells by Western blotting of signaling proteins and cell proliferation and apoptosis assays. Primary AML cells, LAM, and AML tissues expressed CCR3, CXCR4, CXCR6, and CXC3CR1. In AML cells, their ligands CXCL12 CX3CL1, CCL11, CCL24, and CCL28 caused robust phosphorylation of p42/44 MAPK and Akt. CXCL12 was expressed in type II pneumocytes covering LAM nodules and caused AML cell growth and protection from apoptosis, which was blocked by AMD3100, a CXCR4 inhibitor. The mTOR inhibitor rapamycin, but not AMD3100, inhibited growth of AML tumor xenografts. We conclude that the CXCL12/CXCR4 axis promotes, but is not absolutely required for, AML/LAM cell growth and survival.
Assuntos
Angiomiolipoma/imunologia , Angiomiolipoma/patologia , Quimiocina CXCL12/fisiologia , Linfangioleiomiomatose/imunologia , Linfangioleiomiomatose/patologia , Receptores CXCR4/fisiologia , Angiomiolipoma/metabolismo , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Linhagem Celular Transformada , Movimento Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/metabolismo , Feminino , Humanos , Mediadores da Inflamação/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/metabolismo , Camundongos , Camundongos Nus , Receptores CXCR4/biossíntese , Receptores CXCR4/metabolismo , Transdução de Sinais/imunologia , Proteína 1 do Complexo Esclerose Tuberosa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Matrix metalloproteinases (MMPs) have been implicated in lung cyst formation in lymphangioleiomyomatosis (LAM). As doxycycline inhibits MMP activity in vivo, some patients take doxycycline, as one report has suggested a possible benefit in LAM. However, there have been no randomized controlled clinical trials of doxycycline for LAM, and any mechanism of action is unclear. Here, we examine previously proposed mechanisms of actions. Cell proliferation and adhesion were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and Cytomatrix cell adhesion kits. Apoptosis was examined by TdT-mediated dUTP nick end labeling (TUNEL) assay. MMP-2 expression was examined by quantitative real-time PCR and zymography in doxycycline-treated ELT3 cells and tumor growth using angiomyolipoma-derived tumor xenografts in nude mice. In ELT3 cells, >or=25 microg/ml doxycycline decreased proliferation, increased apoptosis, and caused a change in cell morphology associated with redistribution of actin stress filaments. Reduction in proliferation was also seen in human angiomyolipoma-derived cells. Cell adhesion to ECM proteins was decreased by doxycycline at 50 microg/ml and prevented detachment of already adherent cells. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In the xenograft model, doxycycline (30 mg*kg(-1)*day(-1)) had no effect on tumor growth, final tumor weight, or tumor lysate MMP levels. Doxycycline at doses >or= 25 microg/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. Doxycycline had no effect on MMP-2 expression or activity or tumor growth in the xenograft model. Any possible in vivo effect is unlikely to be mediated by MMP-2 or reduced cell proliferation.
Assuntos
Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxiciclina/administração & dosagem , Linfangioleiomiomatose/patologia , Linfangioleiomiomatose/fisiopatologia , Metaloproteinases da Matriz/biossíntese , Inibidores de Proteases/administração & dosagem , Angiomiolipoma/metabolismo , Angiomiolipoma/patologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ratos , Transplante HeterólogoRESUMO
Angiomyolipomas are benign mesenchymal tumours of smooth muscle, blood vessels and fat which occur sporadically or associated with tuberous sclerosis and lymphangioleiomyomatosis (LAM), a rare cystic lung disease. Angiomyolipoma and LAM are caused by loss of function of either the tuberous sclerosis-1 or -2 genes resulting in activation of p70S6kinase (S6K1) and uncontrolled cellular proliferation. LAM and angiomyolipoma can be exacerbated by oestrogens but how this occurs is not understood. To address this question, we created a xenograft tumour system in nude mice using immortalised angiomyolipoma cells. Angiomyolipoma xenografts had active S6K1, p38, p42/44 MAPK and Akt; they grew more rapidly and had greater Akt phosphorylation after oestrogen treatment of tumour-bearing mice. Transcriptional profiling showed oestrogen induced 300 genes including extracellular matrix proteins, proteases, cell cycle regulatory proteins and growth factors including platelet derived growth factor-C (PDGF-C). Biologically active PDGF-C was produced by primary angiomyolipoma cells in culture and PDGF-C protein was present in the neoplastic smooth muscle cells of 5/5 human angiomyolipoma and 4/5 LAM tissues examined by immunohistochemistry. These findings suggest that the response to oestrogen in this model is mediated by activation of Akt and transcriptional events. This model may prove useful for studying the biology and effect of drugs on angiomyolipoma and diseases related to TSC.
Assuntos
Angiomiolipoma/metabolismo , Estrogênios/farmacologia , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Angiomiolipoma/genética , Angiomiolipoma/fisiopatologia , Animais , Linhagem Celular Transformada , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/metabolismo , Linfangioleiomiomatose/fisiopatologia , Linfocinas/genética , Linfocinas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Complexos Multiproteicos , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17alpha(1) is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17alpha(1) gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-beta signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17alpha(1) gene.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/metabolismo , Regiões Promotoras Genéticas , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Blástula/embriologia , Blástula/metabolismo , Proteínas de Ligação a DNA/genética , Endoderma/embriologia , Endoderma/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Mutação , Fatores de Transcrição SOXF , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
A comprehensive Arabidopsis genomic resource has been developed at the Nottingham Arabidopsis Stock Centre (NASC) to support the international plant community. This browser, termed AtEnsembl, provides a detailed and user-friendly interface for accessing a wide range of Arabidopsis-based genomic information and post-genomic resources using the Ensembl browser. The resource aims to provide the broadest possible range of Ensembl features, including pointers to germplasm as well as representations of gene and protein information, links to Affymetrix gene expression data, and extensive data download capabilities.
Assuntos
Arabidopsis/genética , Bases de Dados Genéticas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Genômica/métodos , Armazenamento e Recuperação da Informação/métodos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin (TSC1 and 2), form a heteromer, which represses the kinase mammalian target of rapamycin. Loss of TSC1 or 2 results in diseases characterized by loss of cell-cycle control, including TSC and lymphangioleiomyomatosis. As tuberin has multiple signaling inputs, including phosphatidylinositide-3-OH kinase, mitogen-activated protein kinase, and adenosine monophosphate kinase, we postulated tuberin would have multiple protein interactions governed by subcellular localization and cellular status and examined this in primary human airway smooth muscle cells. Using immunofluorescence and confocal microscopy, tuberin was detected in cytoplasm, nucleus, nucleoli, and mitochondria. Fractionation of synchronized airway smooth cells showed that tuberin enters the nucleus in late G(1), and passage through the cell cycle is necessary for nuclear entry. Deletion constructs showed localization sequences for the nucleus between amino acids 1351 and 1807, for mitochondria between 901 and 1350, and for cytoplasmic speckles between 1 and 450. Using fluorophore-tagged proteins, we observed fluorescence resonance energy transfer between tuberin and hamartin within these speckles, indicating a direct interaction between the proteins at this site. The observations that tuberin is localized to mitochondria and translocated to the nucleus in G(1) are novel and consistent with interactions with proteins within multiple signaling pathways. Dynamic relocalization of tuberin may control these interactions to integrate these pathways. As tuberin has potential roles in proliferation, migration, and cell phenotype, it therefore warrants further investigation in diseases categorized by abnormalities in airway smooth muscle.
Assuntos
Músculo Liso/citologia , Músculo Liso/metabolismo , Traqueia/citologia , Traqueia/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular , Ciclo Celular , Células Cultivadas , Fase G1 , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
The maternal determinant VegT is required for both endoderm and mesoderm formation by the Xenopus embryo. An important downstream mediator of VegT action is Xsox17, which has been proposed to be induced in cell-autonomous, then signal-dependent phases. We show that Xsox17 is a direct VegT target, but that direct induction of Xsox17 by VegT is rapidly inhibited. This inhibition is relieved by TGF- beta signalling, to which the future endoderm cell is sensitised by VegT, resulting in the observed dependence on cell contact for maintained Xsox17 expression. We propose that this change in regulation is a consequence of a VegT-induced repressor, inhibiting direct induction of early endoderm markers by VegT, and contributing to the formation of the boundary of the endodermal domain.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Grupo de Alta Mobilidade , Proteínas com Domínio T/fisiologia , Fatores de Transcrição , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Xenopus , Xenopus/embriologia , Animais , Indução Embrionária , Endoderma/citologia , Endoderma/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Modelos Biológicos , Proteínas/genética , Proteínas/fisiologia , Fatores de Transcrição SOXF , Transdução de Sinais , Proteínas com Domínio T/genética , Xenopus/genética , Xenopus/fisiologiaRESUMO
We have used antisense morpholino oligos to establish the developmental roles of three Xsox17 proteins in Xenopus development (Xsox17alpha(1), alpha(2) and beta). We show that their synthesis can be inhibited with modest amounts of oligo. The inhibition of each individually produces defects in late midgut development. Loss of activity of the Xsox17alpha proteins additionally inhibits hindgut formation, and inhibiting Xsox17alpha(1) disrupts foregut development with variable penetrance. When all Xsox17 activity is inhibited cell movements are halted during late gastrulation and the transcription of several endodermally expressed genes is reduced. Thus the Xsox17 proteins have redundant roles in early development of the endoderm and partly distinct roles during later organogenesis.