RESUMO
The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.
Assuntos
Receptor alfa de Estrogênio , Insulina , Animais , Feminino , Masculino , Camundongos , Células Endoteliais , Glucose , Músculo Esquelético , Receptores de EstrogênioRESUMO
Activation of the Hedgehog (Hh) signaling pathway by mutations within its components drives the growth of several cancers. However, the role of Hh pathway activation in lung cancers has been controversial. Here, we demonstrate that the canonical Hh signaling pathway is activated in lung stroma by Hh ligands secreted from transformed lung epithelia. Genetic deletion of Shh, the primary Hh ligand expressed in the lung, in KrasG12D/+;Trp53fl/fl autochthonous murine lung adenocarcinoma had no effect on survival. Early abrogation of the pathway by an anti-SHH/IHH antibody 5E1 led to significantly worse survival with increased tumor and metastatic burden. Loss of IHH, another Hh ligand, by in vivo CRISPR led to more aggressive tumor growth suggesting that IHH, rather than SHH, activates the pathway in stroma to drive its tumor suppressive effects-a novel role for IHH in the lung. Tumors from mice treated with 5E1 had decreased blood vessel density and increased DNA damage suggestive of reactive oxygen species (ROS) activity. Treatment of KrasG12D/+;Trp53fl/fl mice with 5E1 and N-acetylcysteine, as a ROS scavenger, decreased tumor DNA damage, inhibited tumor growth and prolonged mouse survival. Thus, IHH induces stromal activation of the canonical Hh signaling pathway to suppress tumor growth and metastases, in part, by limiting ROS activity.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Proteínas Hedgehog/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Acetilcisteína/farmacologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Mutação/genética , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.
Assuntos
Divisão Celular , Túbulos Renais/embriologia , Túbulos Renais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Células Cultivadas , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Doenças Renais Císticas/patologia , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Morfogênese , Néfrons/metabolismo , Néfrons/patologia , Fuso Acromático/metabolismoRESUMO
Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulin(GFP)), we discover that α-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 µm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.
Assuntos
Medula Óssea/anatomia & histologia , Células-Tronco Hematopoéticas/metabolismo , Imagem Molecular , Animais , Arteríolas/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Divisão Celular , Linhagem da Célula , Quimiocina CXCL12/metabolismo , Diáfises/citologia , Diáfises/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores para Leptina/metabolismo , Nicho de Células-Tronco , Tíbia/anatomia & histologia , Tíbia/irrigação sanguínea , Tíbia/citologia , alfa Catenina/análise , alfa Catenina/metabolismoAssuntos
Portadores de Fármacos/química , Micelas , Nanopartículas/química , Aminas , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Endossomos/metabolismo , Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lisossomos/metabolismo , Espectroscopia de Ressonância Magnética , Polietilenoglicóis/química , Rodaminas , Relação Estrutura-AtividadeRESUMO
WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.
Assuntos
Citocinese/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Análise de Variância , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Citocinese/genética , Citoplasma/metabolismo , Imunofluorescência , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HT29 , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos de Histocompatibilidade Menor , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Ratos , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Intraflagellar transport (IFT) of a approximately 17S particle containing at least 16 distinct polypeptides is required for the assembly and maintenance of cilia and flagella. Although both genetic and biochemical evidence suggest a role for IFT in vertebrate photoreceptors, the spatial distribution of IFT proteins within photoreceptors remains poorly defined. We have evaluated the distribution of 4 IFT proteins using a combination of immunocytochemistry and rod-specific overexpression of GFP tagged IFT proteins. Endogenous IFT proteins are most highly concentrated within the inner segment, around the basal body, and within the outer segment IFT proteins are localized in discrete particles along the entire length of the axoneme. IFT52-GFP and IFT57-GFP mimicked this pattern in transgenic Xenopus.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/metabolismo , Camundongos , Retina/diagnóstico por imagem , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Ultrassonografia , XenopusRESUMO
The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.
Assuntos
Biomarcadores/metabolismo , Mesoderma/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Actinas/metabolismo , Animais , Aquaporina 5 , Aquaporinas/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fibroblastos/patologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/metabolismo , Fenótipo , Alvéolos Pulmonares/citologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1RESUMO
Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cílios/química , Proteínas do Citoesqueleto/química , Flagelos/química , Proteínas Musculares/química , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Western Blotting , Bovinos , Proteínas do Citoesqueleto/metabolismo , Dimerização , Hidrólise , Íons , Rim/metabolismo , Cinesinas , Masculino , Camundongos , Modelos Biológicos , Células Fotorreceptoras/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Testículo/metabolismo , Técnicas do Sistema de Duplo-Híbrido , alfa-Galactosidase/metabolismoAssuntos
Cílios , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/fisiopatologia , Sensação , Animais , Transporte Biológico , Movimento Celular , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/metabolismo , Camundongos , Camundongos Mutantes/genética , Mutação , Células Fotorreceptoras de Vertebrados/patologia , Isoformas de Proteínas/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
Cytoplasmic dynein is involved in a wide variety of cellular functions. In addition to the initially characterized form (MAP 1C/dynein 1), a second form of cytoplasmic dynein (dynein 2) has been identified and implicated in intraflagellar transport (IFT) in lower eukaryotes and in Golgi organization in vertebrates. In the current study, the primary structure of the full-length dynein 2 heavy chain (HC) was determined from cDNA sequence. The dynein 1 and dynein 2 sequences were similar within the motor region, and around the light intermediate chain (LIC)-binding site within the N-terminal stem region. The dynein 2 HC co-immunoprecipitated with LIC3, a homologue of dynein 1 LICs. Dynein 2 mRNA was abundant in the ependymal layer of the neural tube and in the olfactory epithelium. Antibodies to dynein 2 HC, LIC3 and a component of IFT particles strongly stained the ependymal layer lining the lateral ventricles. Both dynein 2 HC and LIC3 staining was also observed associated with connecting cilia in the retina and within primary cilia of non-neuronal cultured cells. These data support a specific role for dynein 2 in the generation and maintenance of cilia.