RESUMEN
Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17,181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease.
Asunto(s)
Ancirinas/genética , Mutación , Regiones Promotoras Genéticas , Esferocitosis Hereditaria/genética , Factor de Transcripción TFIID/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Genoma Humano , Humanos , Células K562 , Sitio de Iniciación de la TranscripciónRESUMEN
The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5' untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.
Asunto(s)
Ancirinas/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Sitios de Unión/genética , Sistema Libre de Células , Secuencia de Consenso , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Biblioteca de Genes , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries. METHODS: Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France). RESULTS: Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression. CONCLUSION: The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.
Asunto(s)
Flavonoides/farmacología , Fenoles/farmacología , Túnica Media/fisiología , Vasoconstricción/efectos de los fármacos , Vino , Bradiquinina/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Histamina/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfolipasas A/metabolismo , Polifenoles , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Quinacrina/farmacología , Ingeniería de Tejidos , Túnica Media/efectos de los fármacos , Túnica Media/metabolismo , Vasoconstricción/fisiología , Vasodilatadores/farmacología , Quinasas Asociadas a rhoRESUMEN
We have developed a tissue-engineering approach for the production of a completely biological blood vessel from cultured human cells. In the present study, we took advantage of this tissue-engineering method to demonstrate that it can be used to reproduce the subtle differences in the expression of receptors present on the media of native human blood vessels. Indeed, a small percentage (3 of 18) of native human umbilical cord veins (HUCVs) responded to endothelin, the most powerful vasopressor agent known to date, via both endothelin A (ET(A)) and endothelin B (ET(B)) receptor activation. In contrast, most HUCVs tested responded to ET via ET(A) receptor activation only. Tissue-engineered vascular media (TEVM) were next reconstructed by using vascular smooth muscle cells (VSMCs) isolated and cultured from HUCVs expressing both ET(A) and ET(B) receptors to determine the functional integrity of our TEVM model. The reconstructed TEVM presents an endothelin response similar to that of respective HUCVs from which VSMCs were isolated. Reverse transcriptase polymerase chain reaction on TEVM reconstructed in vitro correlated these vasocontractile profiles by showing the presence of messenger RNA for both ET(A) and ET(B) receptors. Taken together with recently published results on TEVM expressing only ET(A) receptor, these results show that our reconstructed TEVM present a similar ET response profile as the blood vessel from which the VSMCs were isolated and cultured. These findings indicate that subtle differences, such as receptor expression, are preserved in the reconstructed tissue. Therefore, our TEVM offers a valuable human in vitro model with which to study the functionality of human blood vessels, such as their vasoactive response, or to perform pharmacologic studies.
Asunto(s)
Vasos Sanguíneos/metabolismo , Medios de Cultivo , Técnicas de Cultivo de Tejidos , Ingeniería de Tejidos , Endotelinas/fisiología , Humanos , Venas Umbilicales/metabolismoRESUMEN
Whether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ET(A) receptors, whereas that of the media and the adventitia+media was ET(A) and ET(B) receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.
Asunto(s)
Prótesis Vascular , Tejido Conectivo/fisiología , Vasoconstricción , Ácido Aspártico Endopeptidasas/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiología , Enzimas Convertidoras de Endotelina , Endotelinas/farmacología , Humanos , Metaloendopeptidasas/metabolismo , ARN Mensajero/metabolismo , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Ingeniería de Tejidos , Túnica Media/metabolismo , Túnica Media/fisiología , Vasoconstricción/efectos de los fármacos , VasodilataciónRESUMEN
BACKGROUND: Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor's hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel. METHODS AND RESULTS: Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ET(A) receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ET(A) but not the ET(B) receptor was present in these human tissue-engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media. CONCLUSIONS: Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ET(A) receptors and endothelin-converting enzyme.
Asunto(s)
Endotelinas/fisiología , Receptor de Endotelina A/fisiología , Ingeniería de Tejidos , Túnica Media/fisiología , Ácido Aspártico Endopeptidasas/metabolismo , Células Cultivadas/citología , Células Endoteliales/citología , Enzimas Convertidoras de Endotelina , Endotelio Vascular/citología , Humanos , Metaloendopeptidasas/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor de Endotelina A/biosíntesis , Receptor de Endotelina A/genética , Receptor de Endotelina B/biosíntesis , Receptor de Endotelina B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túnica Media/efectos de los fármacos , Venas Umbilicales/citología , Resistencia Vascular , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Progress in tissue engineering now allows the recreation of functional blood vessels from cultured human vascular cells. When reconstructed under specific conditions, their structure, mechanical properties and function (especially vasomotricity) allow them to be used as human models for studying the biology and pharmacology of blood vessels. These models may help to circumvent the limitations in the obtention and use of native human blood vessels for experimental purpose.
Asunto(s)
Vasos Sanguíneos , Ingeniería de Tejidos , Investigación Biomédica , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Humanos , Ingeniería de Tejidos/métodos , Vasoconstricción , VasodilataciónRESUMEN
The inositol 1,4,5-trisphosphate receptor (InsP3R) is a ligand-gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti-InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS-7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co-localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity-binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed.