RESUMEN
Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor ß (TGF-ß) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-ß. The results revealed that Smad4 inhibition activated interleukin-1ß (IL-1ß) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1ß antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-ß signaling, such as those driven by SMAD4 mutations.
Asunto(s)
Aneurisma de la Aorta/prevención & control , Interleucina-1beta/antagonistas & inhibidores , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Cultivadas , Quimiocina CCL2/antagonistas & inhibidores , Interleucina-1beta/biosíntesis , Ratones , Miocitos del Músculo Liso/inmunología , FN-kappa B/fisiología , Receptores CCR2/antagonistas & inhibidores , Proteína Smad4/fisiología , Tamoxifeno/farmacologíaRESUMEN
Objective- EMILIN-1 (elastin microfibrils interface located protein-1) protein inhibits pro-TGF-ß (transforming growth factor-ß) proteolysis and limits TGF-ß bioavailability in vascular extracellular matrix. Emilin1-/- null mice display increased vascular TGF-ß signaling and are hypertensive. Because EMILIN-1 is expressed in vessels from embryonic life to adulthood, we aimed at unravelling whether the hypertensive phenotype of Emilin1-/- null mice results from a developmental defect or lack of homeostatic role in the adult. Approach and Results- By using a conditional gene targeting inactivating EMILIN-1 in smooth muscle cells of adult mice, we show that increased blood pressure in mice with selective smooth muscle cell ablation of EMILIN-1 depends on enhanced myogenic tone. Mechanistically, we unveil that higher TGF-ß signaling in smooth muscle cells stimulates HB-EGF (heparin-binding epidermal growth factor) expression and subsequent transactivation of EGFR (epidermal growth factor receptor). With increasing intraluminal pressure in resistance arteries, the cross talk established by TGF-ß and EGFR signals recruits TRPC6 (TRP [transient receptor potential] classical type 6) and TRPM4 (TRP melastatin type 4) channels, lastly stimulating voltage-dependent calcium channels and potentiating myogenic tone. We found reduced EMILIN-1 and enhanced myogenic tone, dependent on increased TGF-ß-EGFR signaling, in resistance arteries from hypertensive patients. Conclusions- Taken together, our findings implicate an unexpected role of the TGF-ß-EGFR pathway in hypertension with current translational perspectives.
Asunto(s)
Receptores ErbB/metabolismo , Hipertensión/metabolismo , Glicoproteínas de Membrana/metabolismo , Arterias Mesentéricas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Vasoconstricción , Animales , Presión Sanguínea , Canales de Calcio/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Canales Catiónicos TRPM/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
OBJECTIVE: Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-ß processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. METHODS AND RESULTS: This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1(-/-) background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1(-/-) mice. We also explored the effect of treatment with anti-TGF-ß antibodies on the hypertensive phenotype of Emilin1(-/-) mice, finding that neutralization of TGF-ß in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-ß activity. CONCLUSIONS: Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-ß.
Asunto(s)
Presión Sanguínea , Hipertensión/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Vasoconstricción , Animales , Anticuerpos Neutralizantes/administración & dosificación , Arteriolas/metabolismo , Arteriolas/fisiopatología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Monitoreo Ambulatorio de la Presión Arterial/métodos , Relación Dosis-Respuesta a Droga , Ecocardiografía Doppler , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Genotipo , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo , Telemetría , Factores de Tiempo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Vasoconstrictores/farmacologíaRESUMEN
Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.
Asunto(s)
Apoptosis , Colágeno Tipo VI/deficiencia , Mitocondrias Musculares/patología , Enfermedades Mitocondriales/patología , Enfermedades Musculares/patología , Animales , Calcio/metabolismo , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Homocigoto , Inmunosupresores/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Oligomicinas/farmacología , Retículo Sarcoplasmático/ultraestructuraRESUMEN
Collagen VI is a survival factor for skeletal muscle produced by endomysial cells and localized in connective tissue around muscle fibers. Mutations of its genes (COL6A1, COL6A2 and COL6A3) cause two muscular disorders, Bethlem myopathy and Ullrich disease. Expression of Collagen VI is highly dynamic during development, suggesting that developmental and homeostatic cues of the muscle microenvironment are relevant to confine its expression in this tissue. In face of the large body of work highlighting the relevance for human diseases of the adhesion of muscle cells with their surrounding extracellular matrix, remarkably little is known on how myogenic cells control gene expression in the connective tissue cells that produce such matrix. By expressing promoter-lacZ constructs in transgenic mice, we identify a Col6a1 gene enhancer region that is necessary for activation of transcription in connective tissue cells associated with skeletal muscle. By means of a lacZ transgenic mouse line crossed in metD/D mutant background, in which muscles of limb buds fail to form, we provide evidence that the presence of cells of the myogenic lineage is needed for enhancer activation in mesenchymal cells. Accordingly, lack of myogenic cells in limb buds of metD/D mice reduces Collagen VI deposition in connective tissue. The Col6a1 enhancer characterized here is conserved in mammals and may be relevant in some cases of heritable diseases of Collagen VI.
Asunto(s)
Colágeno Tipo VI/genética , Células del Tejido Conectivo/metabolismo , Elementos de Facilitación Genéticos/genética , Células Musculares/metabolismo , Activación Transcripcional , Animales , Colágeno Tipo VI/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Transducción de SeñalRESUMEN
EMILINs constitute a family of genes of the extracellular matrix with high structural similarity. Four genes have been identified so far in human and mouse. To gain insight into the function of this gene family, EMILIN-1 has been inactivated in the mouse by gene targeting. The homozygous animals were fertile and did not show obvious abnormalities. However, histological and ultrastructural examination revealed alterations of elastic fibers in aorta and skin. Formation of elastic fibers by mutant embryonic fibroblasts in culture was also abnormal. Additional alterations were observed in cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae. Considering that EMILIN-1 is adhesive for cells and that the protein binds to elastin and fibulin-5, EMILIN-1 may regulate elastogenesis and vascular cell maintenance by stabilizing molecular interactions between elastic fiber components and by endowing elastic fibers with specific cell adhesion properties.
Asunto(s)
Vasos Sanguíneos/anomalías , Tejido Elástico/anomalías , Proteínas de la Matriz Extracelular/deficiencia , Glicoproteínas de Membrana/deficiencia , Animales , Vasos Sanguíneos/patología , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Tejido Elástico/patología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/fisiología , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía ElectrónicaRESUMEN
Aortic valve disease (AVD) is a common condition with a progressive natural history, and presently, there are no pharmacologic treatment strategies. Elastic fiber fragmentation (EFF) is a hallmark of AVD, and increasing evidence implicates developmental elastic fiber assembly defects. Emilin1 is a glycoprotein necessary for elastic fiber assembly that is present in both developing and mature human and mouse aortic valves. The Emilin1-deficient mouse (Emilin1-/- ) is a model of latent AVD, characterized by activated TGFß/MEK/p-Erk signaling and upregulated elastase activity. Emilin1-/- aortic valves demonstrate early EFF and aberrant angiogenesis followed by late neovascularization and fibrosis. The objective of this study was to test the effectiveness of three different targeted therapies. Aged (12-14 months) Emilin1-/- mice were treated with refametinib (RDEA-119, MEK1/2 inhibitor), doxycycline (elastase inhibitor), or G6-31 (anti-VEGF-A mouse antibody) for 4 weeks. Refametinib- and doxycycline-treated Emilin1-/- mice markedly reduced MEK/p-Erk activation in valve tissue. Furthermore, both refametinib and doxycycline attenuated elastolytic cathepsin K, L, MMP-2, and MMP-9 activation, and abrogated macrophage and neutrophil infiltration in Emilin1-/- aortic valves. RNAseq analysis was performed in aortic valve tissue from adult (4 months) and aged (14 months) Emilin1-/- and age-matched wild-type control mice, and demonstrated upregulation of genes associated with MAPK/MEK/p-Erk signaling and elastases at the adult stage and inflammatory pathways at the aged stage controlling for age. These results suggest that Erk1/2 signaling is an important modulator of early elastase activation, and pharmacological inhibition using refametinib may be a promising treatment to halt AVD progression.
Asunto(s)
Anticuerpos/uso terapéutico , Válvula Aórtica/efectos de los fármacos , Difenilamina/análogos & derivados , Doxiciclina/uso terapéutico , Cardiopatías Congénitas/tratamiento farmacológico , Enfermedades de las Válvulas Cardíacas/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glicoproteínas de Membrana/genética , Sulfonamidas/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos/farmacología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Enfermedad de la Válvula Aórtica Bicúspide , Difenilamina/farmacología , Difenilamina/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Doxiciclina/farmacología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Ratones , Ratones Noqueados , Elastasa Pancreática/metabolismo , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Expression of EMILIN-1, the first member of a newly discovered family of extracellular matrix genes, has been investigated during mouse development. EMILIN-1 mRNA is detectable in morula and blastocyst by RT-PCR. First expression of the gene is found by in situ hybridization in ectoplacental cone in embryos of 6.5 days and in extraembryonic visceral endoderm at 7.5 days. The allantois is also labeled. Staining of ectoplacental cone-derived secondary trophoblast giant cells and spongiotrophoblast is strong up to 11.5 days and then declines. In the embryo, high levels of mRNA are initially expressed in blood vessels, perineural mesenchyme and somites at 8.5 days. Later on, intense labeling is identified in the mesenchymal component of organs anlage (i.e. lung and liver) and different mesenchymal condensations (i.e. limb bud and branchial arches). At late gestation staining is widely distributed in interstitial connective tissue and smooth muscle cell-rich tissues. The data suggest that EMILIN-1 may have a function in placenta formation and initial organogenesis and a later role in interstitial connective tissue.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Ratones/embriología , Animales , Northern Blotting , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Hibridación in Situ , Glicoproteínas de Membrana/genética , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
The EDEN gene superfamily comprises genes that contain the EMI domain, a structural motif recently identified in proteins of the extracellular matrix. We report here the detailed expression pattern of genes of the EMILIN/Multimerin family, the most numerous group of EDEN superfamily, during mouse development. In situ hybridization has revealed that the EMILIN/Multimerin genes are particularly expressed in the cardio-vascular system and in mesenchymal cells. In general, the territories of expression of each gene are partially overlapping or complementary with that of other members of the family and, usually, more than one gene of the family is active in different tissues, consistent with the possibility of functional compensation. The analysis is particularly relevant in the interpretation of gene targeting experiments.
Asunto(s)
Animales Recién Nacidos/metabolismo , Proteínas Sanguíneas/metabolismo , Embrión de Mamíferos/metabolismo , Glicoproteínas de Membrana/metabolismo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Proteínas Sanguíneas/genética , Sistema Cardiovascular/embriología , Sistema Cardiovascular/metabolismo , Desarrollo Embrionario y Fetal , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Glicoproteínas de Membrana/genética , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Familia de Multigenes , Estructura Terciaria de Proteína , Terminología como Asunto , Distribución TisularRESUMEN
Aortic valve disease (AVD) is characterized by elastic fiber fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is an elastin-binding glycoprotein that regulates elastogenesis and inhibits TGF-ß signaling, but the role of Emilin1 in valve tissue is unknown. We tested the hypothesis that Emilin1 deficiency results in AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-ß dysregulation. Using histology, immunohistochemistry, electron microscopy, quantitative gene expression analysis, immunoblotting and echocardiography, we examined the effects of Emilin1 deficiency (Emilin1-/-) in mouse aortic valve tissue. Emilin1 deficiency results in early postnatal cell-matrix defects in aortic valve tissue, including EFF, that progress to latent AVD and premature death. The Emilin1-/- aortic valve displays early aberrant provisional angiogenesis and late neovascularization. In addition, Emilin1-/- aortic valves are characterized by early valve interstitial cell activation and proliferation and late myofibroblast-like cell activation and fibrosis. Interestingly, canonical TGF-ß signaling (phosphorylated Smad2 and Smad3) is upregulated constitutively from birth to senescence, whereas non-canonical TGF-ß signaling (phosphorylated Erk1 and Erk2) progressively increases over time. Emilin1 deficiency recapitulates human fibrotic AVD, and advanced disease is mediated by non-canonical (MAPK/phosphorylated Erk1 and Erk2) TGF-ß activation. The early manifestation of EFF and aberrant angiogenesis suggests that these processes are crucial intermediate factors involved in disease progression and therefore might provide new therapeutic targets for human AVD.
Asunto(s)
Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Glicoproteínas de Membrana/deficiencia , Neovascularización Patológica/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Válvula Aórtica/ultraestructura , Enfermedad de la Válvula Aórtica Bicúspide , Calcinosis/complicaciones , Calcinosis/patología , Proliferación Celular , Cutis Laxo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Tejido Elástico/metabolismo , Fibrosis , Cardiopatías Congénitas/complicaciones , Cardiopatías Congénitas/diagnóstico por imagen , Enfermedades de las Válvulas Cardíacas/complicaciones , Enfermedades de las Válvulas Cardíacas/diagnóstico por imagen , Inflamación/complicaciones , Inflamación/patología , Glicoproteínas de Membrana/metabolismo , Ratones , Modelos Biológicos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Neovascularización Patológica/patología , Transducción de Señal , UltrasonografíaRESUMEN
RNA interference (RNAi) through the use of lentiviral vectors is a valuable technique to induce loss of function mutations in mammals. Although very promising, the method has found only limited application and its general applicability remains to be established. Here we analyze how different factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue specific expression. Our results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of insertion of the provirus. Although low copy number may produce efficient interference with low frequency, the general trend is that the number of integrated proviral copies determines the level of silencing and the severity of phenotypic traits. The site of insertion not only determines the overall intensity of expression of the small interfering RNA (siRNA), but also introduces slight variability of silencing in different organs. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline-inducible production of siRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of siRNA synthesis was not complete. The data support the application of lentiviral vectors used here in transgenesis.
Asunto(s)
Colágeno Tipo VI/metabolismo , Lentivirus/genética , Interferencia de ARN , Animales , Apoptosis , Northern Blotting , Línea Celular , Colágeno Tipo VI/genética , Femenino , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Immunoblotting , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Transgénicos , Células 3T3 NIH , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.
Asunto(s)
Regulación de la Expresión Génica , Linfangiogénesis , Vasos Linfáticos/metabolismo , Glicoproteínas de Membrana/fisiología , Animales , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Vasos Linfáticos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , FenotipoRESUMEN
TGF-beta proteins are main regulators of blood vessel development and maintenance. Here, we report an unprecedented link between TGF-beta signaling and arterial hypertension based on the analysis of mice mutant for Emilin1, a cysteine-rich secreted glycoprotein expressed in the vascular tree. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced vessel size. Mechanistically, we found that Emilin1 inhibits TGF-beta signaling by binding specifically to the proTGF-beta precursor and preventing its maturation by furin convertases in the extracellular space. In support of these findings, genetic inactivation of Emilin1 causes increased TGF-beta signaling in the vascular wall. Strikingly, high blood pressure observed in Emilin1 mutants is rescued to normal levels upon inactivation of a single TGF-beta1 allele. This study highlights the importance of modulation of TGF-beta availability in the pathogenesis of hypertension.
Asunto(s)
Presión Sanguínea/fisiología , Homeostasis , Glicoproteínas de Membrana/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Arterias/citología , Arterias/metabolismo , Furina/metabolismo , Dosificación de Gen , Genes Reporteros , Humanos , Hipertensión/etiología , Hipertensión/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Nodal , Fenotipo , Precursores de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The location of regions that regulate transcription of the murine Emilin1 gene was investigated in a DNA fragment of 16.8 kb, including the entire gene and about 8.7 and 0.6 kb of 5'- and 3'-flanking sequences, respectively. The 8.7-kb segment contains the 5'-end of the putative 2310015E02Rik gene and the sequence that separates it from Emilin1, whereas the 0.6-kb fragment covers the region between Emilin1 and Ketohexokinase genes. Sequence comparison between species identified several conserved regions in the 5'-flanking sequence. Most of them contained chromatin DNase I-hypersensitive sites, which were located at about -950 (HS1), -3100 (HS2), -4750 (HS3), and -5150 (HS4) in cells expressing Emilin1 mRNA. Emilin1 transcription initiates at multiple sites, the major of which correspond to two Initiator sequences. Promoter assays suggest that core promoter activity was mainly dependent on Initiator1 and on Sp1-binding sites close to the Initiators. Moreover, one important regulatory region was contained between -1 and -169 bp and a second one between -630 bp and -1.1 kb. The latter harbors a putative binding site for transcription factor AP1 matching the location of HS1. The function of different regions was studied by expressing lacZ constructs in transgenic mice. The results show that the 16.8-kb segment contains regulatory sequences driving high level transcription in all the tissues where Emilin1 is expressed. Moreover, the data suggest that transcription in different tissues is achieved through combinatorial cooperation between various regions, rather than being dependent on a single cis-activating region specific for each tissue.
Asunto(s)
Regulación de la Expresión Génica , Genes Reguladores , Glicoproteínas de Membrana/genética , Transcripción Genética , Región de Flanqueo 3' , Región de Flanqueo 5' , Animales , Secuencia de Bases , Genes Reporteros , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
Collagen VI is a main extracellular matrix protein whose mutation is linked to myopathic diseases. In myoblasts and other cell types, collagen VI gene transcription peaks during cell-cycle exit that precedes differentiation, upon serum withdrawal or confluence. To get insight into this transcriptional regulation, we characterized a growth arrest responsive region (GARR) in the Col6a1 promoter responsible for this effect. In this work, we identify sterol regulatory element binding protein (SREBP) as a GARR binding protein and provide evidence that SREBP contributes to induction of Col6a1 transcription in serum free conditions. Furthermore, our data unveil a previously unexpected link between extracellular matrix production and LDL signaling.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Colágeno Tipo VI/biosíntesis , Proteínas de Unión al ADN/fisiología , Factores de Transcripción , Transcripción Genética , Animales , Northern Blotting , Núcleo Celular/metabolismo , Colágeno Tipo VI/genética , Medio de Cultivo Libre de Suero/farmacología , ADN Complementario/metabolismo , Regulación de la Expresión Génica , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Células Musculares/metabolismo , Células 3T3 NIH , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
Wntbeta-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated beta-catenin during mammalian development, we generated the beta-catenin-activated transgene driving expression of nuclear beta-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of beta-cateninT cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of beta-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wntbeta-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia+) background revealed betacatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wntbeta-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteínas de Pez Cebra , Animales , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Hibridación in Situ , Operón Lac , Ratones , Ratones Transgénicos , Mutación , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Transactivadores/genética , Proteínas Wnt , beta CateninaRESUMEN
HMGA2(2) belongs to the high mobility group A (HMGA) family of architectural transcription factors which participate in a wide variety of nuclear processes ranging from transcription to recombination, playing an important role in chromatin remodelling. HMGA2 is expressed during embryogenesis but not by adult somatic tissues, yet it becomes re-expressed following neoplastic transformation. A role in development is underscored by the finding that the inactivation of the Hmga2 gene is responsible for the murine pygmy phenotype. To elucidate mechanisms that control HMGA2 expression, we have previously cloned the gene and identified functional elements involved in its regulation. In this paper, transgenic mice were generated to define genomic regions involved in Hmga2 developmental and tissue-specific transcriptional regulation. A genomic region from -8.1 to -3.7kb upstream from the initiation site has been found to recapitulate most of the spatial and temporal endogenous Hmga2 gene expression.