RESUMO
Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Lesão Pulmonar , Animais , Camundongos , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Lesão Pulmonar/patologia , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.
Immune cells in the lung help guard against infections. On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria. When the immune cells detect these invaders, the TLR receptors spring into action and trigger an inflammatory response to destroy the microbes. This inflammation usually helps the lung clear infections. But it can also be harmful and damage the lung, for example when inflammation is caused by non-infectious substances such as pollutants in the atmosphere. There are several TLR receptors that each recognize a specific molecule. In 2010, researchers showed that the receptor TLR4 is responsible for causing inflammation in the lung after exposure to pollution. Another receptor called TLR5 also helps activate the immune response in the lung. But it was unclear whether this receptor also plays a role in pollution-linked lung damage. Now, Hussain, Johnson, Sciurba et al. including one of the researchers involved in the 2010 study have investigated the role of TLR5 in immune cells from the lungs of humans and mice. The experiments showed that TLR5 works together with TLR4 and helps trigger an inflammatory response to both pollutants and bacteria. Hussain et al. found that people lacking a working TLR5 receptor (which make up 310% of the population) are less likely to experience lung inflammation when exposed to pollution or bacterial proteins that activate TLR4. These findings suggest that people without TLR5 may be protected from pollution-induced lung injury. Further research into the role of TLR5 could help develop genetic tests for identifying people who are more sensitive to damage from pollution. This information could then be used to determine the likelihood of a patient experiencing certain lung diseases.
Assuntos
Lesão Pulmonar , Fator 88 de Diferenciação Mieloide , Transdução de Sinais , Receptor 4 Toll-Like , Receptor 5 Toll-Like , Animais , Células Cultivadas , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/metabolismoRESUMO
We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.
Assuntos
Células da Medula Óssea/patologia , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Neoplasias Cutâneas/patologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Proteína Morfogenética Óssea 5/metabolismo , Movimento Celular , Plasticidade Celular/fisiologia , Técnicas de Cocultura , Células Epiteliais/citologia , Feminino , Proteína HMGB1/metabolismo , Folículo Piloso/citologia , Queratinócitos/patologia , Queratinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/patologia , Papiloma/patologia , Células-Tronco/citologia , Células-Tronco/patologia , Acetato de Tetradecanoilforbol/toxicidade , Células Tumorais CultivadasRESUMO
Allergic asthma is a major cause of morbidity in both pediatric and adult patients. Recent research has highlighted the role of hyaluronan (HA), an extracellular matrix glycosaminoglycan, in asthma pathogenesis. Experimental allergic airway inflammation and clinical asthma are associated with an increase of shorter fragments of HA (sHA), which complex with inter-α-inhibitor heavy chains (HCs) and induce inflammation and airway hyperresponsiveness (AHR). Importantly, the effects of sHA can be antagonized by the physiological counterpart high molecular weight HA (HMWHA). We used a mouse model of house dust mite-induced allergic airway inflammation and demonstrated that instilled HMWHA ameliorated allergic airway inflammation and AHR, even when given after the establishment of allergic sensitization and after challenge exposures. Furthermore, instilled HMWHA reduced the development of HA-HC complexes and the activation of Rho-associated, coiled-coil containing protein kinase 2. We conclude that airway application of HMWHA is a potential treatment for allergic airway inflammation.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Modelos Animais de Doenças , Ácido Hialurônico/administração & dosagem , Inflamação/prevenção & controle , Pyroglyphidae/patogenicidade , Hipersensibilidade Respiratória/prevenção & controle , Animais , Feminino , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Hipersensibilidade Respiratória/etiologiaRESUMO
Acid (HCl) aspiration during anesthesia may lead to acute lung injury. There is no effective therapy. We hypothesized that HCl instilled intratracheally in C57BL/6 mice results in the formation of low-molecular weight hyaluronan (L-HA), which activates RhoA and Rho kinase (ROCK), causing airway hyperresponsiveness (AHR) and increased permeability. Furthermore, instillation of high-molecular weight hyaluronan (H-HA; Yabro) will reverse lung injury. We instilled HCl in C57BL/6 wild-type (WT), myeloperoxidase gene-deficient (MPO-/-) mice, and CD44 gene-deficient (CD44-/-) mice. WT mice were also instilled intranasally with H-HA (Yabro) at 1 and 23 h post-HCl. All measurements were performed at 1, 5, or 24 h post-HCl. Instillation of HCl in WT but not in CD44-/- resulted in increased inflammation, AHR, lung injury, and L-HA in the bronchoalveolar lavage fluid (BALF) 24 h post-HCl; L-HA levels and lung injury were significantly lower in HCl-instilled MPO-/- mice. Isolated perfused lungs of HCl instilled WT but not of CD44-/- mice had elevated values of the filtration coefficient ( Kf). Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer decreased barrier resistance ( RT). H-HA significantly mitigated inflammation, AHR, and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT, as well as ROCK2 phosphorylation. Increased H- and L-HA levels were found in the BALF of mechanically ventilated patients but not in healthy volunteers. HCl instillation-induced lung injury is mediated by the L-HA-CD44-RhoA-ROCK2 signaling pathway, and H-HA is a potential novel therapeutic agent for acid aspiration-induced lung injury.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Barreira Alveolocapilar/efeitos dos fármacos , Receptores de Hialuronatos/fisiologia , Ácido Hialurônico/farmacologia , Ácido Clorídrico/toxicidade , Peroxidase/fisiologia , Pneumonia/tratamento farmacológico , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Barreira Alveolocapilar/metabolismo , Barreira Alveolocapilar/patologia , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Troca Gasosa Pulmonar , Viscossuplementos/farmacologiaRESUMO
Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.
Assuntos
Asma/enzimologia , Regulação Enzimológica da Expressão Gênica , Hialuronan Sintases/biossíntese , Pulmão/enzimologia , Miócitos de Músculo Liso/enzimologia , Miofibroblastos/enzimologia , Actinas/biossíntese , Actinas/genética , Alérgenos/toxicidade , Animais , Asma/induzido quimicamente , Asma/genética , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/genética , Doença Crônica , Humanos , Hialuronan Sintases/genética , Pulmão/patologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Miofibroblastos/patologiaRESUMO
Primary cilia (PC) are solitary cellular organelles that play critical roles in development, homeostasis, and disease pathogenesis by modulating key signaling pathways such as Sonic Hedgehog and calcium flux. The antenna-like shape of PC enables them also to facilitate sensing of extracellular and mechanical stimuli into the cell, and a critical role for PC has been described for mesenchymal cells such as chondrocytes. However, nothing is known about the role of PC in airway smooth muscle cells (ASMCs) in the context of airway remodeling. We hypothesized that PC on ASMCs mediate cell contraction and are thus integral in the remodeling process. We found that PC are expressed on ASMCs in asthmatic lungs. Using pharmacological and genetic methods, we demonstrated that PC are necessary for ASMC contraction in a collagen gel three-dimensional model both in the absence of external stimulus and in response to the extracellular component hyaluronan. Mechanistically, we demonstrate that the effect of PC on ASMC contraction is, to a small extent, due to their effect on Sonic Hedgehog signaling and, to a larger extent, due to their effect on calcium influx and membrane depolarization. In conclusion, PC are necessary for the development of airway remodeling by mediating calcium flux and Sonic Hedgehog signaling.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Brônquios/patologia , Cílios/patologia , Asma/metabolismo , Asma/patologia , Brônquios/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Cílios/metabolismo , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Potenciais da Membrana/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Transdução de Sinais/fisiologiaRESUMO
The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.
Assuntos
Ferro/metabolismo , Placenta/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Tristetraprolina/genética , Saco Vitelino/metabolismo , Animais , Desenvolvimento Embrionário , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/metabolismo , Tristetraprolina/deficiência , Tristetraprolina/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
The epidermal growth factor receptor (EGFR) is necessary for normal involution of hair follicles after the growth phase of anagen, although the mechanisms through which it acts are not well understood. In this report, we used transcriptional profiling of microdissected hair follicles from mice with skin-targeted deletion of Egfr to investigate how EGFR activation triggers catagen. Immunofluorescence for phospho-EGFR in mouse skin revealed increased activation of EGFR in follicular keratinocytes at catagen onset. Consistent with other models of EGFR deficiency, mice with skin-targeted deletion of Egfr (Krt14-Cre(+) /Egfr(fl/fl) ) exhibited a delayed and asynchronous catagen entry. Transcriptional profiling at the time of normal catagen onset at post-natal day (P) 17 revealed increased expression of the mitotic regulator Rcc2 in hair follicles lacking EGFR. Rcc2 protein was strongly immunopositive in the nuclei of control follicular keratinocytes at P16 then rapidly decreased until it was undetectable between P18 and 21. In contrast, Rcc2 expression continued in Egfr mutant follicles throughout this period. Proliferation, measured by bromodeoxyuridine incorporation, was also significantly increased in Egfr mutant follicular keratinocytes compared to controls at P18-21. Similarly, Rcc2-regulated mitotic regulator Stathmin 1 was strikingly reduced in control but not Egfr mutant follicles between P17 and P19. Deletion of Stmn1, in turn, accelerated catagen entry associated with premature cessation of proliferation in the hair follicles. These data reveal EGFR suppression of mitotic regulators including Rcc2 and Stathmin 1 as a mechanism for catagen induction in mouse skin.
Assuntos
Receptores ErbB/metabolismo , Estatmina/metabolismo , Animais , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Receptores ErbB/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Folículo Piloso/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Transdução de Sinais , Pele/metabolismo , Estatmina/genéticaRESUMO
Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects.
RESUMO
Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.
Assuntos
Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/tratamento farmacológico , Ácido Hialurônico/uso terapêutico , Lesão Pulmonar/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , alfa-Globulinas/antagonistas & inibidores , alfa-Globulinas/biossíntese , alfa-Globulinas/imunologia , Animais , Hiper-Reatividade Brônquica/imunologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio , Canais de Cálcio/metabolismo , Células Cultivadas , Cloro/toxicidade , Ativação Enzimática , Matriz Extracelular , Inflamação , Potenciais da Membrana/efeitos dos fármacos , Cloreto de Metacolina/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo , Traqueia/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness.
Assuntos
Proteínas ADAM/genética , Expressão Gênica/genética , Receptor ErbB-2/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Regulação para Cima/genética , Proteína ADAM12 , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Pele/patologia , Raios Ultravioleta/efeitos adversosRESUMO
Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Fosfopeptídeos/metabolismo , Proteômica , Neoplasias Cutâneas/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Movimento Celular , Transformação Celular Neoplásica , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Humanos , Marcação por Isótopo , Camundongos , Papiloma/metabolismo , Papiloma/patologia , Fosfopeptídeos/análise , Fosforilação , Proteína Quinase C/metabolismo , Proteoma/metabolismo , Pele/metabolismo , Neoplasias Cutâneas/patologia , Espectrometria de Massas em Tandem , Titânio/química , Células Tumorais Cultivadas , Regulação para Cima , Quinases Ativadas por p21/metabolismo , Quinases da Família src/metabolismoRESUMO
The multistage model of nonmelanoma skin carcinogenesis has contributed significantly to our understanding of epithelial cancer in general. We used the Krt1-15CrePR1;R26R transgenic mouse to determine the contribution of keratin 15+ cells from the hair follicle to skin tumor development by following the labeled progeny of the keratin 15 expressing cells into papillomas. We present three novel observations. First, we found that keratin 15 expressing cells contribute to most of the papillomas by 20 weeks of promotion. Second, in contrast to the transient behavior of labeled keratin 15-derived progeny in skin wound healing, keratin 15 progeny persist in papillomas, and some malignancies for many months following transient induction of the reporter gene. Third, papillomas have surprising heterogeneity not only in their cellular composition, but also in their expression of the codon 61 signature Ha-ras mutation with approximately 30% of keratin 15-derived regions expressing the mutation. Together, these results demonstrate that keratin 15 expressing cells of the hair follicle contribute to cutaneous papillomas with long term persistence and a subset of which express the Ha-ras signature mutation characteristic of initiated cells.
Assuntos
Transformação Celular Neoplásica/patologia , Folículo Piloso/patologia , Queratina-15/fisiologia , Papiloma/patologia , Neoplasias Cutâneas/patologia , Células-Tronco/patologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Feminino , Genes ras/genética , Folículo Piloso/efeitos dos fármacos , Humanos , Integrases/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Microdissecção e Captura a Laser , Camundongos , Camundongos Transgênicos , Mutação/genética , Papiloma/induzido quimicamente , Papiloma/genética , Pele/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Células-Tronco/efeitos dos fármacos , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Cell division cycle 25A (CDC25A) is a dual-specificity phosphatase that removes inhibitory phosphates from cyclin-dependent kinases, allowing cell-cycle progression. Activation of cell-cycle checkpoints following DNA damage results in the degradation of CDC25A, leading to cell-cycle arrest. Ultraviolet (UV) irradiation, which causes most skin cancer, results in both DNA damage and CDC25A degradation. We hypothesized that ablation of CDC25A in the skin would increase cell-cycle arrest following UV irradiation, allowing for improved repair of DNA damage and decreased tumorigenesis. Cdc25a(fl/fl) /Krt14-Cre recombinase mice, with decreased CDC25A in the epithelium of the skin, were generated and exposed to UV. UV-induced DNA damage, in the form of cyclopyrimidine dimers and 8-oxo-deoxyguanosine adducts, was eliminated earlier from CDC25A-deficient epidermis. Surprisingly, loss of CDC25A did not alter epidermal proliferation or cell cycle after UV exposure. However, the UV-induced apoptotic response was prolonged in CDC25A-deficient skin. Double labeling of cleaved caspase-3 and the DNA damage marker γH2A.X revealed many of the apoptotic cells in UV-exposed Cdc25a mutant skin had high levels of DNA damage. Induction of skin tumors by UV irradiation of Cdc25a mutant and control mice on a skin tumor susceptible to v-ras(Ha) Tg.AC mouse background revealed UV-induced papillomas in Cdc25a mutants were significantly smaller than in controls in the first 6 weeks following UV exposure, although there was no difference in tumor multiplicity or incidence. Thus, deletion of Cdc25a increased apoptosis and accelerated the elimination of DNA damage following UV but did not substantially alter cell-cycle regulation or tumorigenesis.
Assuntos
Apoptose/efeitos da radiação , Dano ao DNA , DNA/efeitos da radiação , Pele/efeitos da radiação , Fosfatases cdc25/fisiologia , Animais , Ciclo Celular , Proliferação de Células/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Pele/patologia , Raios Ultravioleta , Fosfatases cdc25/deficiênciaRESUMO
Induction of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, in ODC transgenic skin stimulates epidermal proliferation but not hyperplasia, activates underlying stromal cells and promotes skin tumorigenesis following a single subthreshold dose of a carcinogen. Because chronic wounds are a well-recognized risk factor for skin cancer, we investigated the response to a tissue remodeling event in normal skin that is abraded to remove only the epidermal layer in K6/ODC transgenic (follicular ODC expression) and in inducible ODCER transgenic mice (suprabasal ODC expression). When regenerative epidermal hyperplasia was resolved in normal littermates following abrasion, ODC transgenic mice exhibited progressive epidermal hyperplasia with formation of benign tumor growths and maintained an increased epidermal proliferation index and activation of translation-associated proteins at abrasion sites. The epidermal hyperplasia and tumor-like growth was accompanied by activation of underlying stromal cells and prolonged infiltration of inflammatory cells. Treatment with the anti-inflammatory agent dexamethasone did not reduce the high proliferative index in the regenerated epidermis but dramatically reduced the epidermal hyperplasia and prevented the wound-induced tumor growths in abraded ODCER skin. Treatment with α-difluoromethylornithine, a specific inhibitor of ODC activity, normalized the wound response in transgenic mice and decreased wound-induced inflammation if administered from the time of abrasion but not if initiated 4 days following abrasion. These results suggest a role for polyamines in prolonging wound-associated inflammation in addition to stimulating proliferation both of which are sufficient to sustain epidermal hyperplasia and benign tumor growth even in the absence of genetic damage.
Assuntos
Ornitina Descarboxilase/fisiologia , Neoplasias Cutâneas/etiologia , Ferimentos e Lesões/enzimologia , Animais , Poliaminas Biogênicas/fisiologia , Proliferação de Células , Epiderme/patologia , Hiperplasia , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Cutâneas/patologia , Cicatrização , Ferimentos e Lesões/complicaçõesRESUMO
Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.
Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , Queratinócitos/efeitos da radiação , Ceratose Actínica/genética , Neoplasias Induzidas por Radiação/genética , PTEN Fosfo-Hidrolase/fisiologia , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversos , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular/metabolismo , Linhagem Celular/patologia , Linhagem Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Reparo do DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Ceratose Actínica/patologia , Camundongos , Neoplasias Induzidas por Radiação/patologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Papiloma/etiologia , Papiloma/genética , Papiloma/patologia , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/genética , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown.Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309-382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.
RESUMO
The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1-transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage-independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth. © 2011 Wiley Periodicals, Inc.
Assuntos
Neoplasias Cutâneas/patologia , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibição de Contato , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , TransfecçãoRESUMO
Sp proteins are evolutionarily conserved transcription factors required for the expression of a wide variety of genes that are critical for development and cell cycle progression. Deregulated expression of certain Sp proteins is associated with the formation of a variety of human tumors; however, direct evidence that any given Sp protein is oncogenic has been lacking. Here, we report that Sp2 protein abundance in mice increases in concert with the progression of carcinogen-induced murine squamous cell carcinomas. Transgenic mice specifically overexpressing murine Sp2 in epidermal basal keratinocytes were highly susceptible to wound- and carcinogen-induced papillomagenesis. Transgenic animals that were homozygous rather than hemizygous for the Sp2 transgene exhibited a striking arrest in the epidermal differentiation program, perishing within 2 weeks of birth. Our results directly support the likelihood that Sp2 overexpression occurring in various human cancers has significant functional effect.