RESUMO
Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.
Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Enteropatogênica/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Proteínas de Escherichia coli/fisiologia , Fatores de Virulência/fisiologia , Linhagem Celular , Sobrevivência Celular , Humanos , Fibras de Estresse/metabolismo , Junções ÍntimasRESUMO
The hair follicle is an intricate miniature organ dedicated to the production of the structural hair fiber, which is largely composed of hair keratin (HK) proteins. Many developmental pathways contribute to hair follicle development; however, the molecular control of HK genes is still far from being resolved. Because the nuclear factor (NF)-kappaB pathway is known to be involved in the morphogenesis of the hair follicle, we explored the possibility that it may also regulate HK expression. To this end, we analyzed the effect of p65/RelA, an NF-kappaB effector, on HK regulatory regions using transient transfections into tissue culture cells. Reporter assays on cells transfected with HK promoter constructs and real-time polymerase chain reaction analysis of endogenous HK gene activity demonstrated that p65 induces transcriptional activation of several HK genes of human and mouse origin, primarily that of acidic hair keratin 5 (Ha5). Focusing on the highly responsive human Ha5 gene, we defined the major NF-kappaB/RelA binding sites in its regulatory region and showed the direct binding of p65 to these sites using gel shift assays. We further show, using immunohistochemistry on human hair follicle sections, that p65 is co-expressed with HKs in the hair shaft compartment and may thus be the effector that mediates the NF-kappaB pathway's activity, which recently was genetically demonstrated to be active in the same region. Thus, we provide evidence for a previously unknown function of NF-kappaB in hair formation-direct activation of HK target genes-a function that may shed light on some of the symptoms of ectodermal dysplasias.
Assuntos
Folículo Piloso/metabolismo , Queratinas Específicas do Cabelo/genética , Queratinas Tipo II/genética , Queratinas Tipo I/genética , Fator de Transcrição RelA/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas/metabolismo , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinas Específicas do Cabelo/biossíntese , Queratinas Tipo I/biossíntese , Queratinas Tipo II/biossíntese , Camundongos , NF-kappa B/fisiologia , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/fisiologia , Fator de Transcrição RelA/genéticaRESUMO
The three mammalian Runx transcription factors, some of which are known to be involved in human genetic diseases and cancer, are pivotal players in embryo development and function as key regulators of cell fate determination and organogenesis. Here, we report the expression of Runx1 during the development of hair and other skin appendages in the mouse and describe the effect of Runx1 on the structural hair output. In hair follicles, where the three Runx proteins are expressed, Runx1 expression is most prominent in both mesenchymal and epithelial compartments. The epithelial expression includes the hair keratin forming layers of the hair shaft and the bulge, where interestingly, Runx1 is co-expressed with keratin 15, a putative hair follicle stem cell marker. In the hair mesenchyme, during early stages of hair morphogenesis, Runx1 is expressed in a discrete dermal sub-epithelial layer, while at later stages it is found in a hair cycle dependent pattern in the dermal papilla. To elucidate the function of Runx1 in the hair follicle we have generated a Runx1 epidermal conditional knockout and found that the mutant mice display a remarkable structural deformation of the zigzag hair type. The data delineate Runx1 as a novel specific marker of several hair follicle cell types and sheds light on its role in hair morphogenesis and differentiation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Pele/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Cabelo/citologia , Cabelo/embriologia , Camundongos , Camundongos Knockout , Pele/citologia , Pele/embriologia , Pele/crescimento & desenvolvimentoRESUMO
Transcriptional regulators of the Runx family play critical roles in normal organ development and, when mutated, lead to genetic diseases and cancer. Runx3 functions during cell lineage decisions in thymopoiesis and neurogenesis and mediates transforming growth factor-beta signaling in dendritic cells. Here, we study the function of Runx3 in the skin and its appendages, primarily the hair follicle, during mouse development. Runx3 is expressed predominantly in the dermal compartment of the hair follicles as they form and during the hair cycle, as well as in the nail and sweat gland skin appendages. Distinct expression is also detected periodically in isolated cells of the epidermis and in melanocytes, populating the hair bulb. Runx3-deficient mice display a perturbation of the normal hair coat, which we show to be due to hair type and hair shape changes. Thus, one of the functions of Runx3 in skin may be to regulate the formation of the epithelial derived structural hair by affecting dermal to epidermal interactions.