RESUMO
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-beta1. As the ECM critically regulates AEC responses to TGF-beta1, we explored the role of the prominent epithelial integrin alpha3beta1 in experimental fibrosis by generating mice with lung epithelial cell-specific loss of alpha3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through beta-catenin has been implicated in EMT; we found that in primary AECs, alpha3 integrin was required for beta-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-beta-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-beta-catenin/pSmad2 complexes and showed accumulation of pY654-beta-catenin in myofibroblasts. These findings demonstrate epithelial integrin-dependent profibrotic crosstalk between beta-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.
Assuntos
Células Epiteliais/fisiologia , Fibroblastos/metabolismo , Integrina alfa3beta1/metabolismo , Fibrose Pulmonar , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , beta Catenina/metabolismo , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Células Cultivadas , Células Epiteliais/citologia , Fibroblastos/citologia , Humanos , Integrina alfa3beta1/genética , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Transgênicos , Fenótipo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Proteína Smad2/genéticaRESUMO
Cortical GABAergic interneurons, most of which originate in the ganglionic eminences, take distinct tangential migratory trajectories into the developing cerebral cortex. However, the ligand-receptor systems that modulate the tangential migration of distinct groups of interneurons into the emerging cerebral wall remain unclear. Here, we show that netrin-1, a diffusible guidance cue expressed along the migratory routes traversed by GABAergic interneurons, interacts with alpha3beta1 integrin to promote interneuronal migration. In vivo analysis of interneuron-specific alpha3beta1 integrin, netrin-1-deficient mice (alpha3(lox/-)Dlx5/6-CIE, netrin-1(-/-)) reveals specific deficits in the patterns of interneuronal migration along the top of the developing cortical plate, resulting in aberrant interneuronal positioning throughout the cerebral cortex and hippocampus of conditional alpha3(lox/-)Dlx5/6-CIE, netrin-1(-/-) mice. These results indicate that specific guidance mechanisms, such as netrin-1-alpha3beta1 integrin interactions, modulate distinct routes of interneuronal migration and the consequent positioning of groups of cortical interneurons in the developing cerebral cortex.
Assuntos
Movimento Celular , Córtex Cerebral/crescimento & desenvolvimento , Integrina alfa3beta1/metabolismo , Interneurônios/fisiologia , Fatores de Crescimento Neural/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Movimento Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Interneurônios/metabolismo , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Netrina-1 , Proteínas Supressoras de Tumor/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
BACKGROUND: The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer's disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer's disease, have not been examined in depth. METHODS: Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. RESULTS: Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3. CONCLUSIONS: Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer's disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer , Microglia , Proteínas Nucleares , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/metabolismo , Animais , Humanos , Inflamação/metabolismo , Lipopolissacarídeos , Camundongos , Microglia/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteômica , Transcriptoma , Proteínas Supressoras de Tumor/genéticaRESUMO
Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hipóxia/metabolismo , Integrina alfa3beta1/metabolismo , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Citometria de Fluxo , Hipóxia/genética , Hipóxia/patologia , Imuno-Histoquímica , Integrina alfa3beta1/genética , Masculino , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The arachidonic acid metabolite of 12 lipoxygenase, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) promotes metastatic behavior of tumor cells (1). In this study we set out to identify 12(S)-HETE stimulated signaling pathways, and their contribution to cellular functions in A431 epidermoid carcinoma. 1) 12(S)-HETE signaling involves extracellular-regulated protein kinase (ERK1/2), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3 kinase) and Src kinase. 2) 12(S)-HETE stimulates cell migration on laminin, which is eliminated by PKC and PI3 kinase inhibitors, reduced by 50% with Src inhibitor, but unaffected by inhibition of ERK1/2. 3) 12(S)-HETE stimulated spreading on fibronectin relies on ERK1/2 and PI3 kinase activities, but not on PKC or Src. 4) Focal adhesion kinase, a key organizer of focal adhesions, is tyrosine phosphorylated in response of 12(S)-HETE treatment, which requires Src, but not PKC, PI3 kinase or ERK1/2 activity. 5) Inhibition of 12 lipoxygenase leads to apoptosis in serum starved A431 cells. 12(S)-HETE stimulated p90Rsk and Akt, key players in an ERK and a PI3 kinase (respectively) dependent anti apoptotic pathways.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/fisiologia , Transdução de Sinais/fisiologia , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Melanoma , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Células Tumorais CultivadasRESUMO
Pregnancy is associated with transfer of maternal cells to the fetus and fetal cells to the mother. In both cases, the transferred cells are described as microchimeric. Fetal microchimeric cells include semi-allogeneic stem cells, which are few in number and are capable of long-term survival in the "foreign" host. They are recognized by the maternal immune system but not rejected or attacked. These cells appear to survive and even thrive for years in a mother's body, perhaps for her lifetime. Previously regarded as potentially dangerous interlopers that might propagate autoimmune and even malignant disease, fetal microchimeric cells are now increasingly being recognized and analyzed for their healing, reparative, and perhaps regenerative roles. Fetal microchimerism (MC) may make significant and previously unknown positive contributions to women's health, longevity, and risk of disease. This article reviews the history, major discoveries, and current concepts and gaps in knowledge in the field of fetal MC.
Assuntos
Quimerismo , Feto , Saúde da Mulher , Feminino , Humanos , Tolerância Imunológica , GravidezRESUMO
During cutaneous wound healing, epidermal keratinocytes play essential roles in the secretion of factors that promote angiogenesis. However, specific cues in the wound microenvironment that trigger the production of pro-angiogenic factors by keratinocytes, and the cellular receptors that mediate this response, remain unclear. In this study, we exploited a model of conditional integrin knockout to demonstrate impaired wound angiogenesis in mice that lack alpha3beta1 integrin in epidermis. In addition, we used genetic and shRNA approaches to determine that alpha3beta1-integrin deficiency in keratinocytes leads to reduced mRNA and protein expression of the pro-angiogenic factor mitogen-regulated protein 3 (MRP3; also known as PRL2C4), and to demonstrate that this regulation provides a mechanism of keratinocyte-to-endothelial-cell crosstalk that promotes endothelial-cell migration. Finally, we showed that the impaired wound angiogenesis in epidermis-specific alpha3-integrin-knockout mice is correlated with reduced expression of MRP3 in wounded epidermis. These findings identify a novel role for alpha3beta1 integrin in promoting wound angiogenesis through a mechanism of crosstalk from epidermal to endothelial cells, and they implicate MRP3 in this integrin-dependent crosstalk. Such a mechanism represents a novel paradigm for integrin-mediated regulation of wound angiogenesis that extends beyond traditional roles for integrins in cell adhesion and migration.
Assuntos
Proteínas Angiogênicas/metabolismo , Células Endoteliais/metabolismo , Epiderme/fisiologia , Integrina alfa3beta1/metabolismo , Queratinócitos/metabolismo , Neovascularização Fisiológica , Proteínas Angiogênicas/genética , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Células Endoteliais/citologia , Epiderme/patologia , Integrina alfa3beta1/genética , Queratinócitos/citologia , Camundongos , Camundongos Knockout , Ativação Transcricional , Cicatrização/fisiologiaRESUMO
Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. In this study, we demonstrate that alpha3beta1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events.