RESUMO
Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin alphavbeta3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin alphavbeta3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Homeodomínio/fisiologia , Neovascularização Fisiológica/genética , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Divisão Celular/genética , Células Cultivadas , Embrião de Galinha , Ciclina D1/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes Homeobox , Hemangioendotelioma/etiologia , Hemangioendotelioma/genética , Proteínas de Homeodomínio/genética , Humanos , Integrina beta3 , Integrinas/biossíntese , Integrinas/genética , Neovascularização Patológica/genética , Fenótipo , Glicoproteínas da Membrana de Plaquetas/biossíntese , Glicoproteínas da Membrana de Plaquetas/genética , RNA Mensageiro/biossíntese , Fatores de Transcrição , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição , RNA Polimerase I/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/química , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência , Especificidade da Espécie , Proteína de Ligação a TATA-Box , Fatores de Transcrição/química , Transcrição Gênica/genéticaRESUMO
Homeobox-containing genes encode transcriptional regulators involved in cell fate and pattern formation during embryogenesis. Recently, it has become clear that their expression in continuously developing adult tissues, as well as in tumorigenesis, may be of equal importance. In the mouse mammary gland, expression patterns of several homeobox genes suggest a role in epithelial-stromal interactions. Because the stroma and the extracellular matrix (ECM) are known to influence both functional and morphological development of the mammary gland, we asked whether these genes would be expressed postnatally in the gland and also in cell lines in culture and whether they could be modulated by ECM. Using a polymerase chain reaction-base strategy five members of the Hox gene clusters a and b were shown to be expressed in cultured mouse mammary cells. Hoxa-1 and Hoxb-7 were chosen for further analysis. Hoxb-7 was chosen because it had not been described previously in the mammary gland and was modulated at different stages of gland development. Hoxa-1 was chosen because it was reported previously to be expressed only in mammary tumors, and not in normal glands. We showed that culturing the mammary epithelial cell lines SCp2 and CID-9 on a basement membrane (BM) that was previously shown to induce a lactational phenotype was necessary to turn off Hoxb-7, but a change in cell shape, brought about by culturing the cells on an inert substratum such as polyHEMA, was sufficient to downregulate Hoxa-1. This is the first report of modulation of homeobox genes by ECM. The results provide a rationale for the differential pattern of expression in vivo of Hoxa-1 and Hoxb-7 during different stages of development. The culture model should permit further in-depth analysis of the molecular mechanisms involved in how ECM signaling and homeobox genes may interact to bring about tissue organization.