RESUMEN
Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.
Asunto(s)
Plaquetas/efectos de los fármacos , Colágeno/farmacología , Activación Plaquetaria/fisiología , Proteína-Lisina 6-Oxidasa/fisiología , Trombofilia/enzimología , Animales , Plaquetas/citología , Traumatismos de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/etiología , Integrina alfa2beta1/fisiología , Megacariocitos/enzimología , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/farmacología , Adhesividad Plaquetaria/genética , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/genética , Factor Plaquetario 4/genética , Regiones Promotoras Genéticas , Proteína-Lisina 6-Oxidasa/genética , Ratas , Trombofilia/genéticaRESUMEN
Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).
Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Glutámico/toxicidad , Ratones Endogámicos C57BL/fisiología , Mutación/fisiología , Neuronas/fisiología , Proteínas PrPC/biosíntesis , Animales , Células Cultivadas , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas PrPC/genéticaRESUMEN
Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.
Asunto(s)
Proteínas Aviares/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Proteínas Aviares/genética , Células COS , Quinasa de la Caseína II/genética , Células Cultivadas , Embrión de Pollo/embriología , Embrión de Pollo/metabolismo , Pollos , Chlorocebus aethiops , Forminas , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Corazón/embriología , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/genética , Proteínas Musculares/análisis , Proteínas Musculares/genética , Miocitos Cardíacos/citología , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteína Sustrato Asociada a CrK/genética , Genes src/genética , Fosforilación/genética , Animales , Neoplasias de la Mama/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Proteína Sustrato Asociada a CrK/metabolismo , Progresión de la Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Ratones Transgénicos/genética , Ratones Transgénicos/metabolismo , RatasRESUMEN
In normal development and pathology, the vascular system depends on complex interactions between cellular elements, biochemical molecules, and physical forces. The electrokinetic vascular streaming potential (EVSP) is an endogenous extremely low frequency (ELF) electrical field resulting from blood flowing past the vessel wall. While generally unrecognized, it is a ubiquitous electrical biophysical force to which the vascular tree is exposed. Extracellular matrix elastin plays a central role in normal blood vessel function and in the development of atherosclerosis. It was hypothesized that ELF fields of low amplitude would alter elastin accumulation, supporting a link between the EVSP and the biology of vascular smooth muscle cells. Neonatal rat aortic smooth muscle cell cultures were exposed chronically to electrical fields characteristic of the EVSP. Extracellular protein accumulation, DNA content, and electron microscopic (EM) evaluation were performed after 2 weeks of exposure. Stimulated cultures showed no significant change in cellular proliferation as measured by the DNA concentration. The per-DNA normalized protein in the extracellular matrix was unchanged while extracellular elastin accumulation decreased 38% on average. EM analysis showed that the stimulated cells had a 2.85-fold increase in mitochondrial number. These results support the formulation that ELF fields are a potential factor in both normal vessel biology and in the pathogenesis of atherosclerotic diseases including heart disease, stroke, and peripheral vascular disease.
Asunto(s)
Elastina/análisis , Hemorreología/fisiología , Músculo Liso Vascular/citología , Aminoácidos/análisis , Animales , Animales Recién Nacidos , Aorta/citología , Aorta/metabolismo , Aorta/ultraestructura , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de la radiación , Células Cultivadas , ADN/análisis , Campos Electromagnéticos , Fenómenos Electrofisiológicos , Proteínas de la Matriz Extracelular/análisis , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/ultraestructura , Ratas , Ratas Sprague-Dawley , Túnica Media/citología , Túnica Media/metabolismo , Túnica Media/ultraestructura , Resistencia Vascular/fisiologíaRESUMEN
The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor ß (TGFß) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.
Asunto(s)
Colágeno Tipo I/metabolismo , Miocitos del Músculo Liso/fisiología , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Transgenes , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Aorta/citología , Ácido Araquidónico/metabolismo , Bradiquinina/metabolismo , Bradiquinina/farmacología , Células Cultivadas , Colágeno Tipo I/genética , Elastina/genética , Elastina/metabolismo , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ratones , Ratones Transgénicos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/ultraestructura , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Desarrollo Embrionario , Morfogénesis , Animales , Apoptosis , Quinasa de la Caseína II/deficiencia , Proliferación Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Cabeza/embriología , Esbozos de los Miembros/embriología , Ratones , Fenotipo , Somitos/citología , Somitos/enzimología , Cola (estructura animal)/embriologíaRESUMEN
Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.
Asunto(s)
Vasos Sanguíneos/fisiología , Adhesión Celular/fisiología , Inflamación/metabolismo , Receptor de Adenosina A2B/metabolismo , Animales , Vasos Sanguíneos/citología , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea , Citocinas/metabolismo , Selectina E/metabolismo , Femenino , Genes Reporteros , Molécula 1 de Adhesión Intercelular/metabolismo , Rodamiento de Leucocito , Ratones , Ratones Noqueados , Selectina-P/metabolismo , Receptor de Adenosina A2B/genética , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. METHODS: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. RESULTS: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium. CONCLUSIONS: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/deficiencia , Cristalino/metabolismo , Cristalino/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Proteína Sustrato Asociada a CrK/metabolismo , Epitelio/metabolismo , Epitelio/patología , Exones/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Especificidad de Órganos , Adhesión en Parafina , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rotura Espontánea/patología , Serina/metabolismo , Transducción de SeñalRESUMEN
Within the nervous system, heparan sulfate (HS) of the cell surface and extracellular matrix influences developmental, physiologic and pathologic processes. HS is a functionally diverse polysaccharide that employs motifs of sulfate groups to selectively bind and modulate various effector proteins. Specific HS activities are modulated by 3-O-sulfated glucosamine residues, which are generated by a family of seven 3-O-sulfotransferases (3-OSTs). Most isoforms we herein designate as gD-type 3-OSTs because they generate HS(gD+), 3-O-sulfated motifs that bind the gD envelope protein of herpes simplex virus 1 (HSV-1) and thereby mediate viral cellular entry. Certain gD-type isoforms are anticipated to modulate neurobiologic events because a Drosophila gD-type 3-OST is essential for a conserved neurogenic signaling pathway regulated by Notch. Information about 3-OST isoforms expressed in the nervous system of mammals is incomplete. Here, we identify the 3-OST isoforms having properties compatible with their participation in neurobiologic events. We show that 3-OST-2 and 3-OST-4 are principal isoforms of brain. We find these are gD-type enzymes, as they produce products similar to a prototypical gD-type isoform, and they can modify HS to generate receptors for HSV-1 entry into cells. Therefore, 3-OST-2 and 3-OST-4 catalyze modifications similar or identical to those made by the Drosophila gD-type 3-OST that has a role in regulating Notch signaling. We also find that 3-OST-2 and 3-OST-4 are the predominant isoforms expressed in neurons of the trigeminal ganglion, and 3-OST-2/4-type 3-O-sulfated residues occur in this ganglion and in select brain regions. Thus, 3-OST-2 and 3-OST-4 are the major neural gD-type 3-OSTs, and so are prime candidates for participating in HS-dependent neurobiologic events.
Asunto(s)
Sistema Nervioso Central/enzimología , Sistema Nervioso Periférico/enzimología , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/metabolismo , Células CHO , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Cricetinae , Cricetulus , Femenino , Regulación Enzimológica de la Expresión Génica , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas/enzimología , Neuronas/metabolismo , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Sulfotransferasas/genética , Internalización del VirusRESUMEN
To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.
Asunto(s)
Cadmio/toxicidad , Cobre/metabolismo , Contaminantes Ambientales/toxicidad , Fibroblastos/efectos de los fármacos , Pulmón/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Animales , Biomarcadores/metabolismo , Proteínas de Transporte de Catión/metabolismo , Línea Celular , ATPasas Transportadoras de Cobre , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Tolerancia a Medicamentos , Matriz Extracelular , Fibroblastos/metabolismo , Fibroblastos/patología , Glutamato-Cisteína Ligasa/metabolismo , Homeostasis/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Metalotioneína/metabolismo , Chaperonas Moleculares/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Regulación hacia ArribaRESUMEN
Copper (Cu)-dependent lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin stabilizing the extracellular matrix (ECM). Chronic inhalation of cadmium (Cd), a toxic metal, induces emphysema. To probe mechanisms of Cd injury to the lung, we developed Cd-resistant (CdR) cells from rat fetal lung fibroblasts (RFL6) by chronic exposure to CdCl(2) from 1 to 40 microM and further examined their expressions of LO, LO substrates, and Cu-scavenging thiols. Levels of cellular thiols, metallothionein, and glutathione in CdR cells were elevated to 13.0- and 3.2-fold of parental controls, respectively, whereas LO mRNA and protein levels were markedly reduced in these cells, with catalytic activity declining to only 16% of the parental control. A conspicuous 52 kDa species rather then the normal 50 kDa proenzyme appeared in the CdR cell extract but not in the conditioned medium, which was codistributed with the endoplasmic reticulum marker [DiOC5(3)] within the cell, implying the Cd-induced 52 kDa species as a product of an abnormal LO-processing defect in secretion. Addition of Cu into CdR cell cultures enhanced the expression of LO mRNA, protein and catalytic activities reflecting limitation of Cu bioavailability for LO in these cells. With inhibition of LO, CdR cells also displayed downregulation of collagen and elastin, substrates of LO. Restoration of collagen synthesis by exposure of CdR cells to purified LO or Cu suggests that inhibition of LO and limitation of Cu cofactor by Cd, as key phenotype changes, accelerated collagen and elastin damage, a critical event pertinent to emphysema pathogenesis.
Asunto(s)
Cadmio/toxicidad , Fibroblastos/efectos de los fármacos , Proteína-Lisina 6-Oxidasa/metabolismo , Animales , Células Cultivadas , Colágeno Tipo I/metabolismo , Cobre/farmacología , Tolerancia a Medicamentos , Elastina/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Pulmón/efectos de los fármacos , Pulmón/embriología , Pulmón/metabolismo , Metalotioneína/metabolismo , Proteína-Lisina 6-Oxidasa/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Pulmonary emphysema and vessel wall aneurysms are diseases characterized by elastolytic damage to elastin fibers that leads to mechanical failure. To model this, neonatal rat aortic smooth muscle cells were cultured, accumulating an extracellular matrix rich in elastin, and mechanical measurements were made before and during enzymatic digestion of elastin. Specifically, the cells in the cultures were killed with sodium azide, the cultures were lifted from the flask, cut into small strips, and fixed to a computer-controlled lever arm and a force transducer. The strips were subjected to a broadband displacement signal to study the dynamic mechanical properties of the samples. Also, quasi-static stress-strain curves were measured. The dynamic data were fit to a linear viscoelastic model to estimate the tissues' loss (G) and storage (H) modulus coefficients, which were evaluated before and during 30 min of elastase treatment, at which point a failure test was performed. G and H decreased significantly to 30% of their baseline values after 30 min. The failure stress of control samples was approximately 15 times higher than that of the digested samples. Understanding the structure-function relationship of elastin networks and the effects of elastolytic injury on their mechanical properties can lead to the elucidation of the mechanism of elastin fiber failure and evaluation of possible treatments to enhance repair in diseases involving elastolytic injury.
Asunto(s)
Elastina/química , Elastina/fisiología , Matriz Extracelular/química , Matriz Extracelular/fisiología , Músculo Liso Vascular/fisiología , Elastasa Pancreática/química , Animales , Extractos Celulares/química , Células Cultivadas , Elasticidad , Matriz Extracelular/ultraestructura , Músculo Liso Vascular/química , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Lysyl oxidase (LO), a copper-dependent enzyme, plays a critical role in the formation and repair of the extracellular matrix (ECM) by catalyzing the crosslinking of elastin and collagen. To better understand mechanisms of cigarette smoke (CS)-induced emphysema, we examined changes in LO and its substrates, i.e., elastin and collagen type I, the major components of cellular thiols, i.e., metallothionein (MT) and glutathione (GSH), and gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, in cigarette smoke condensate (CSC)-treated rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to CSC decreased levels of LO catalytic activity, mRNA, and protein, i.e., the 46 kDa preproenzyme, the 50 kDa proenzyme and the 32 kDa mature enzyme in a dose-dependent manner. In addition, CSC also inhibited the expression of collagen type I and elastin, substrates of LO and important components of the lung ECM. Meanwhile, cellular thiols including MT and GSH as well as gamma-GCS were markedly upregulated in CSC-treated cells. To evaluate modulation of LO expression by cellular thiols, we further examined the effect of increased levels of GSH on LO expression at protein and catalytic levels. Interestingly, exposure of cells to glutathione monoethyl ester, a GSH delivery system, effectively elevated cellular GSH levels and induced a dose-dependent decrease in levels of the protein species and catalytic activity of LO. These results suggest that upregulation by CSC of cellular thiols may play an important role in the downregulation of LO and subsequently destabilization of the lung ECM in CS-induced emphysema.
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Fibroblastos/efectos de los fármacos , Glutatión/análogos & derivados , Pulmón/efectos de los fármacos , Proteína-Lisina 6-Oxidasa/metabolismo , Humo/efectos adversos , Compuestos de Sulfhidrilo/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Aminoaciltransferasas/metabolismo , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Elastina/metabolismo , Feto , Fibroblastos/enzimología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glutatión/farmacología , Pulmón/citología , Pulmón/embriología , Metalotioneína/metabolismo , Proteína-Lisina 6-Oxidasa/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Humo/análisis , Nicotiana , Regulación hacia ArribaRESUMEN
Lysyl oxidase (LO) catalyzes crosslinking of collagen and elastin essential for maintaining the structural integrity of the lung extracellular matrix (ECM). To understand mechanisms of cigarette smoke (CS)-induced emphysema, we investigated effects of cigarette smoke condensate (CSC), the particulate matter of CS, on LO mRNA expression in cultured rat fetal lung fibroblasts (RFL6). Exposure of RFL6 cells to 0-120 microg CSC/ml for 24 h induced a dose-dependent inhibition of LO steady-state mRNAs, for example, reducing transcript levels to below 10% of the control in cells incubated with 80-120 microg CSC/ml. Nuclear run-on assays indicated a marked reduction in LO relative transcriptional rates amounting to 27.7% of the control in cells treated with 120 microg CSC/ml. The actinomycin D-chase assay showed that CSC enhanced the instability of LO transcripts. The t1/2 for LO mRNA decay was decreased from 24 h in the control to 4.5 h in cells treated with 120 microg CSC/ml. Moreover, 80-120 microg CSC/ml also inhibited LO promoter activity as revealed by suppression of reporter gene expression in cells transfected with LO promoter-luciferase vectors. Thus, inhibition of LO transcription initiation and enhancement of LO mRNA instability both contributed to downregulation of LO steady-state mRNA in CSC-treated cells. Note that inhibition of LO mRNA expression by CSC was closely accompanied by markedly decreased levels of transcripts of collagen type I and tropoelastin, two substrates of LO. Thus, transcriptional perturbation of LO and its substrates may be a critical mechanism for ECM damage in CS-induced emphysema.
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Pulmón/enzimología , Nicotiana , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Humo/efectos adversos , Animales , Supervivencia Celular , Células Cultivadas , Colágeno Tipo I/genética , Elastina/genética , Fibroblastos/enzimología , Regiones Promotoras Genéticas , Proteína-Lisina 6-Oxidasa/genética , Estabilidad del ARN , ARN Mensajero/análisis , Ratas , Transcripción GenéticaRESUMEN
Androgens are deemed to be critical for the development, growth, and maintenance of penile tissue as well as for erectile function. Androgens are also reported to inhibit differentiation of stroma progenitor cells into adipocytes and promote differentiation into smooth muscle. The objective of this study was to investigate whether androgen deprivation results in accumulation of adipocytes in the corpus cavernosum. Mature, New Zealand white male rabbits were subjected to sham surgery (control) or orchiectomy. Two weeks after surgery, erectile function was assessed by monitoring changes in intracavernosal blood pressure (ICP) in response to pelvic nerve stimulation. All ICP measurements were normalized to the mean systemic arterial blood pressure. In parallel studies, penile cross sections from control and orchiectomized rabbits were fixed and stained with either Masson's trichrome or hematoxylin and eosin to assess smooth muscle and connective tissue content. Alternatively, tissue sections were stained with Toluidine blue to assess accumulation of fat-containing cells. Orchiectomy resulted in loss of erectile function and penile atrophy, associated with reduced trabecular smooth muscle and increased connective tissue content. Most strikingly, tissue from orchiectomized animals exhibited accumulation of fat-containing cells (adipocytes) in the subtunical region of the corpus cavernosum. We hypothesize that androgen deprivation promotes differentiation of progenitor stroma cells into an adipogenic lineage producing fat-containing cells, thus altering erectile function.
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Adipocitos/patología , Andrógenos/deficiencia , Orquiectomía/efectos adversos , Pene/patología , Enfermedades Vasculares/fisiopatología , Tejido Adiposo/patología , Animales , Presión Sanguínea , Masculino , Conejos , Testosterona/sangreRESUMEN
OBJECTIVE: The function of B-Myb, a negative regulator of vascular smooth muscle cell (SMC) matrix gene transcription, was analyzed in the vasculature. METHODS AND RESULTS: Mice were generated in which the human B-myb gene was driven by the basal cytomegalovirus promoter, and 3 founders were identified. Mice appeared to develop normally, and human B-myb was expressed in the aortas. Total B-Myb levels were elevated in aortas of adult transgenic versus wild-type (WT) animals and varied inversely with alpha1(I) collagen mRNA expression. However, neonatal WT and transgenic aortas displayed comparable levels of alpha1(I) collagen mRNA, likely resulting from elevated levels of cyclin A, which ablated repression by B-Myb. Aortic SMCs from adult transgenic animals displayed decreased alpha1(I) collagen mRNA levels. To examine the role of B-Myb after vascular injury, animals were subjected to femoral artery denudation, which induces SMC-rich lesion formation. A dramatic reduction in neointima formation and lumenal narrowing was observed in arteries of B-myb transgenic versus WT mice 4 weeks after injury. CONCLUSIONS: Data indicate that B-Myb, which inhibits matrix gene expression in the adult vessel wall, reduces neointima formation after vascular injury. To analyze B-Myb function in the vasculature, mice overexpressing B-myb were generated. Neonates displayed normal alpha1(I) collagen mRNA levels, whereas adults expressed decreased collagen mRNA in aortas and isolated vascular SMCs. On femoral artery denudation, neointima formation was dramatically reduced in B-myb transgenic mice.
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Proteínas de Ciclo Celular/fisiología , Colágeno/biosíntesis , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transactivadores/fisiología , Túnica Íntima/patología , Factores de Edad , Animales , Animales Recién Nacidos , Aorta/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Colágeno/genética , Ciclina A/biosíntesis , Ciclina A/genética , Citomegalovirus/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Arteria Femoral/lesiones , Arteria Femoral/metabolismo , Arteria Femoral/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transactivadores/biosíntesis , Transactivadores/genética , TransgenesRESUMEN
Sites of A3 adenosine receptor gene expression have not been fully explored nor has this gene's promoter activity been confirmed in vivo. Transgenic mice were generated in which 2.3 kb upstream of the transcriptional start site of the mouse A3 adenosine receptor was coupled to a beta-galactosidase reporter gene. Selective transgene expression was detected in testis and brain as well as at other sites in which A3 adenosine receptor message has not been previously reported, including retinal ganglion cells and smooth muscle cells of the cerebrospinal vasculature. Our study suggests that this promoter may be useful in the selective targeting of gene expression to specific tissues.
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Regiones Promotoras Genéticas , Receptores Purinérgicos P1/genética , Animales , Encéfalo/metabolismo , Femenino , Regulación de la Expresión Génica , Hibridación in Situ , Operón Lac , Masculino , Ratones , Ratones Transgénicos , Modelos Genéticos , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A3 , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismo , Transgenes , beta-Galactosidasa/metabolismoRESUMEN
Mucins are high molecular weight glycoproteins secreted by salivary glands and epithelial cells lining the digestive, respiratory, and reproductive tracts. These glycoproteins, encoded in at least 13 distinct human genes, can be subdivided into gel-forming and membrane-associated forms. The gel-forming mucin MUC5B is secreted by mucous acinar cells in major and minor salivary glands, but little is known about the expression pattern of membrane-associated mucins. In this study, RT-PCR and Northern blotting demonstrated the presence of transcripts for MUC1 and MUC4 in both parotid and submandibular glands, and in situ hybridization localized these transcripts to epithelial cells lining striated and excretory ducts and in some serous acinar cells. The same cellular distribution was observed by immunohistochemistry. Soluble forms of both mucins were detected in parotid secretion after immunoprecipitation with mucin-specific antibodies. These studies have shown that membrane-associated mucins are produced in both parotid and submandibular glands and that they are expressed in different cell types than gel-forming mucins. Although the function of these mucins in the oral cavity remains to be elucidated, it is possible that they both contribute to the epithelial protective mucin layer and act as receptors initiating one or more intracellular signal transduction pathways.
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Proteínas de la Membrana/metabolismo , Mucina-1/metabolismo , Mucinas/metabolismo , Glándula Parótida/metabolismo , Glándula Sublingual/metabolismo , Glándula Submandibular/metabolismo , Northern Blotting , Western Blotting , Humanos , Inmunohistoquímica , Hibridación in Situ , Mucina-1/genética , Mucina 4 , Mucinas/genética , Pruebas de Precipitina , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 µM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.