RESUMO
Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung.
Assuntos
Colágeno/metabolismo , Gelatinases/metabolismo , Pulmão/patologia , Proteínas de Membrana/metabolismo , Fibrose Pulmonar/patologia , Serina Endopeptidases/metabolismo , Animais , Células Cultivadas , Endopeptidases , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatinases/genética , Deleção de Genes , Humanos , Pulmão/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , RNA Mensageiro/genética , Serina Endopeptidases/genética , Regulação para CimaRESUMO
BACKGROUND: Cigarette smoke suppresses innate immunity, making smokers more susceptible to infection. The NLRP3 inflammasome is a multi-protein complex that releases interleukin (IL) -1ß and IL -18. These cytokines are critical for a timely host response to pathogens. Whether cigarette smoke affects NLRP3 protein levels, and its ability to form an inflammasome, is not known. METHODS AND RESULTS: Using the human monocyte THP1 cell line and C57BL/6 mice, we show that cigarette smoke decreases NLRP3 levels in cells by increasing ubiquitin-mediated proteasomal processing. Half-life of NLRP3 is shortened with the exposure to cigarette smoke extract. Cigarette smoke extract reduces cellular NLRP3 protein abundance in the presence of lipopolysaccharide, a known inducer of NLRP3 protein, thereby decreasing the formation of NLRP3 inflammasomes. The release of IL-1ß and IL-18 by inflammasome activation is also decreased with the exposure to cigarette smoke extract both in THP1 cells and primary human peripheral blood macrophages. CONCLUSIONS: Cigarette smoke extract decreased NLRP3 protein abundance via increased ubiquitin-mediated proteasomal processing. The release of IL-1ß and IL-18 is also decreased with cigarette smoke extract. Our findings may provide mechanistic insights on immunosuppression in smokers and unique opportunities to develop a strategy to modulate immune function.
Assuntos
Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Ubiquitinação/imunologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1ß and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1ß in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1ß and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention.
Assuntos
Proteínas de Transporte/imunologia , Proteínas F-Box/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas F-Box/metabolismo , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteólise , Proteínas Ligases SKP Culina F-Box/imunologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.
Assuntos
Apoptose , Proteínas F-Box/metabolismo , Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Trifosfato de Adenosina/metabolismo , Animais , Metabolismo Energético , Proteínas F-Box/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Mutação , Estrutura Secundária de Proteína , Proteínas Repressoras/metabolismo , SurvivinaRESUMO
The clinical utility of the proliferation marker Ki67 in breast cancer treatment and prognosis is an active area of research. Studies have suggested that differences in pre-analytic and analytic factors contribute to low analytical validity of the assay, with scoring methods accounting for a large proportion of this variability. Use of standard scoring methods is limited, in part due to the time intensive nature of such reporting protocols. Therefore, use of digital image analysis tools may help to both standardize reporting and improve workflow. In this study, digital image analysis was utilized to quantify Ki67 indices in 280 breast biopsy and resection specimens during routine clinical practice. The supervised Ki67 indices were then assessed for agreement with a manual count of 500 tumor cells. Agreement was excellent, with an intraclass correlation coefficient of 0.96 for the pathologist-supervised analysis. This study illustrates an example of a rapid, accurate workflow for implementation of digital image analysis in Ki67 scoring in breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Antígeno Ki-67 , Processamento de Imagem Assistida por Computador/métodos , Diagnóstico por Imagem , Projetos de Pesquisa , Biomarcadores Tumorais/análiseRESUMO
INTRODUCTION: Morphologic and molecular data for staging of multifocal lung squamous cell carcinomas (LSCCs) are limited. In this study, whole exome sequencing (WES) was used as the gold standard to determine whether multifocal LSCC represented separate primary lung cancers (SPLCs) or intrapulmonary metastases (IPMs). Genomic profiles were compared with the comprehensive morphologic assessment. METHODS: WES was performed on 20 tumor pairs of multifocal LSCC and matched normal lymph nodes using the Illumina NovaSeq6000 S4-Xp (Illumina, San Diego, CA). WES clonal and subclonal analysis data were compared with histologic assessment by 16 thoracic pathologists. In addition, the immune gene profiling of the study cases was characterized by the HTG EdgeSeq Precision Immuno-Oncology Panel. RESULTS: By WES data, 11 cases were classified as SPLC and seven cases as IPM. Two cases were technically suboptimal. Analysis revealed marked genomic and immunogenic heterogeneity, but immune gene expression profiles highly correlated with mutation profiles. Tumors classified as IPM have a large number of shared mutations (ranging from 33.5% to 80.7%). The agreement between individual morphologic assessments for each case and WES was 58.3%. One case was unanimously interpreted morphologically as IPM and was in agreement with WES. In a further 17 cases, the number of pathologists whose morphologic interpretation was in agreement with WES ranged from two (one case) to 15 pathologists (one case) per case. Pathologists showed a fair interobserver agreement in the morphologic staging of multiple LSCCs, with an overall kappa of 0.232. CONCLUSIONS: Staging of multifocal LSCC based on morphologic assessment is unreliable. Comprehensive genomic analyses should be adopted for the staging of multifocal LSCC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Genômica , Pulmão/patologiaRESUMO
Environmental media discourse analysis allows researchers to explore thematic patterns in media coverage of environmental issues through qualitative observations and quantitative coding. In this paper, we review newspaper media coverage in Florida pertaining to the connections between agriculture, other non-point pollution sources, and downstream ecosystem health. We analyzed 930 articles from 2007 through 2019 to determine common patterns in how these issues are covered by Florida media, including patterns in the causes of environmental problems, potential solutions, and stakeholders mentioned. Findings suggest that water quality problems are often framed as unpredictable or episodic rather than chronic, and as requiring (and only potentially being remediated by) state-level centralized technological or infrastructural solutions. Stakeholder relationships are framed as adversarial, with environmentalists and the agricultural sector as opponents. This case study highlights key power dynamics, barriers, and opportunities for better managing and communicating about agricultural land use practices and associated downstream environmental impacts.
Assuntos
Meios de Comunicação , Ecossistema , Florida , Meio Ambiente , AgriculturaRESUMO
Expansion of α-smooth muscle actin (α-SMA)-expressing airway smooth muscle of the large airways in asthma is well-studied. However, the contribution of α-SMA-expressing cells in the more distal alveolated parenchyma, including pericytes and myofibroblasts within the alveolar septum, to asthma pathophysiology remains relatively unexplored. The objective of this study was to evaluate α-SMA expression in the alveolated parenchyma of individuals with severe asthma (SA), compared with healthy controls or individuals with chronic obstructive pulmonary disease. Using quantitative digital image analysis and video-assisted thoracoscopic surgery lung biopsies, we show that alveolated parenchyma α-SMA expression is markedly reduced in SA in comparison to healthy controls (mean %positive pixels: 12% vs. 23%, P=0.005). Chronic obstructive pulmonary disease cases showed a similar, but trending, decrease in α-SMA positivity compared with controls (mean %positivity: 17% vs. 23%, P=0.107), which may suggest loss of α-SMA expression is a commonality of obstructive lung diseases. The SA group had similar staining for ETS-related gene protein, a specific endothelial marker, comparatively to controls (mean %positive nuclei: 34% vs. 42%, P=0.218), which suggests intact capillary endothelium and likely intact capillary-associated, α-SMA-positive pericytes. These findings suggest that the loss of α-SMA expression in SA may be because of changes in myofibroblast α-SMA expression or cell number. Further study is necessary to fully evaluate possible mechanisms and consequences of this phenomenon.
Assuntos
Actinas/biossíntese , Asma , Regulação da Expressão Gênica , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Alvéolos Pulmonares , Adulto , Idoso , Asma/metabolismo , Asma/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Índice de Gravidade de DoençaRESUMO
OBJECTIVES: Staging of non-small cell lung carcinoma associated with scar is not discussed in detail in the current American Joint Committee on Cancer staging manual. The recommendation is to include the scar area in the tumor size measurement unless the tumor represents a small focus at the edge of the scar. The aim of this study is to investigate if subtraction of the size of the central scar from the total gross size of surgically resected peripheral clinical stage I non-small cell lung carcinoma improves patient stratification into more accurate prognostic groups. MATERIALS AND METHODS: Hematoxylin and eosin sections of 148 non-small cell lung carcinomas (98 adenocarcinomas and 50 squamous cell carcinomas) were reviewed, including 44 adenocarcinomas and 9 squamous cell carcinomas with scar and 54 adenocarcinomas and 41 squamous cell carcinomas without scar. The microscopic size of the invasive tumor component was determined after the average percentage of scar tissue was subtracted from the grossly measured tumor diameter. Manual results were compared to digital image analysis. RESULTS: Adenocarcinoma with scar were associated with better overall (80.5 % vs. 63.2 %, p = 0.026) and cancer specific survival (95.2 % vs. 73.3 %, p = 0.0053) when compared to adenocarcinoma without scar. Better cancer specific survival was observed in acinar and papillary predominant adenocarcinoma (95.8 % with scar vs. 67.8 % without scar, p = 0.01); while similar trend although not statistically significant was observed in adenocarcinomas with solid or micropapillary component. Using microscopic size, pathologic T stage was down-staged in 21 adenocarcinomas. Squamous cell carcinoma with or without scar did not show a difference in survival. Manual and quantitative image analysis showed strong correlation (r = 0.9769, p < 0.0001). CONCLUSION: Our study suggests that microscopic size of the invasive component in acinar and papillary predominant adenocarcinoma with scar might be a better predictor of survival than the total gross size.
Assuntos
Adenocarcinoma de Pulmão/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Cicatriz/patologia , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma de Células Escamosas/cirurgia , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
BACKGROUND: A number of ancillary tests have been developed that aid in the diagnosis of mesothelioma in cytology specimens. The aim of this retrospective study was to determine whether testing for BAP1 and CDKN2A/p16 status in effusion specimens preceding the tissue diagnosis of mesothelioma would improve diagnostic accuracy and allow an earlier diagnosis of malignancy. METHODS: The study cohort included 99 matched cytology fluid specimens from 74 patients with a surgical specimen diagnosis of malignant mesothelioma (67 epithelioid, 7 biphasic, 55 pleural, and 19 peritoneal). BAP1 immunohistochemistry and p16 fluorescence in situ hybridization (FISH) were performed retrospectively. RESULTS: BAP1 or p16 FISH testing revealed a loss in 7 of 18 (39%) samples originally categorized as benign/reactive, 20 of 33 (61%) interpretable samples categorized as atypical, and 10 of 14 (71%) cases suspicious for mesothelioma. In some cases, the diagnosis of mesothelioma could have been made up to 9 months before biopsy. Similarly, loss of BAP1 or p16 was found in 28 of 30 (93%) samples categorized as malignant, with some cases diagnosable up to 6 months before biopsy. Overall, loss of BAP1 and/or CDKN2A/p16 homozygous deletion would change the diagnostic interpretation in 37 of 60 (62%) (P = .07) effusion specimens, particularly in pleural effusions (32 of 48 samples) (P = .002). The sensitivity of morphologic interpretation alone was 30.3%; however, adding testing for BAP1 and p16 resulted in an increase in sensitivity to 68.7%. (P < .0001). CONCLUSION: These findings suggest that routine use of BAP1 immunochemistry and p16 FISH as adjunctive tests improves the diagnostic accuracy of cytology specimens and potentially allows an earlier diagnosis of malignant mesothelioma.
Assuntos
Biomarcadores Tumorais/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Citodiagnóstico/métodos , Mesotelioma Maligno/diagnóstico , Derrame Pleural/patologia , Deleção de Sequência , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Testes Genéticos/métodos , Homozigoto , Humanos , Masculino , Mesotelioma Maligno/genética , Mesotelioma Maligno/cirurgia , Pessoa de Meia-Idade , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/cirurgia , Derrame Pleural/genética , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/genética , Neoplasias Pleurais/cirurgia , Prognóstico , Estudos Retrospectivos , Adulto JovemRESUMO
Interleukin- (IL-) 22 signaling is protective in animal models of pneumonia and bacteremia by Klebsiella pneumoniae and mediates tissue recovery from influenza and Staph aureus infection. We recently described processing of mouse lung epithelial IL-22 receptor (IL-22R) by ubiquitination on the intracellular C-terminal. To identify cellular factors that regulate human IL-22R, we screened receptor abundance while overexpressing constituents of the ubiquitin system and identify that IL-22R can be shuttled for degradation by multiple previously uncharacterized F-box protein E3 ligase subunits. We observe that in human cells IL-22R is destabilized by FBXW12. FBXW12 causes depletion of endogenous and plasmid-derived IL-22R in lung epithelia, binds the E3 ligase constituent Skp-1, and facilitates ubiquitination of IL-22R in vitro. FBXW12 knockdown with shRNA increases IL-22R abundance and STAT3 phosphorylation in response to IL-22 cytokine treatment. FBXW12 shRNA increases human epithelial cell growth and cell cycle progression with enhanced constitutive activity of map kinases JNK and ERK. These findings indicate that the heretofore-undescribed protein FBXW12 functions as an E3 ligase constituent to ubiquitinate and degrade IL-22R and that therapeutic FBXW12 inhibition may enhance IL-22 signaling and bolster mucosal host defense and infection containment.