RESUMEN
Angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R) signaling. However, the precise molecular mechanism of AT2R-mediated AT1R inhibition remains poorly understood. Here, we characterized the local and functional interaction of AT2R with AT1R. AT2R colocalized and formed a complex with AT1R at the plasma membrane, even in the absence of AII. Upon AII stimulation, the spatial arrangement of the complex was modulated, as confirmed by Förster resonance energy transfer (FRET) analysis, followed by AT2R internalization along with AT1R. AT2R internalization was specifically observed only in the presence of AT1R; AT2R alone could not be internalized. The AT1R-specific inhibitor losartan completely inhibited both the conformational change and the internalization of AT2R with AT1R, whereas the AT2R-specific inhibitor PD123319 partially hindered these phenomena, demonstrating that the activation of both receptors was indispensable for these effects. In addition, treatment with the protein kinase C (PKC) inhibitors inhibited the ligand-dependent accumulation of AT2R but not that of AT1R in the endosomes. A mutation in the putative phosphorylation sites of AT2R also abrogated the co-internalization of ATR2 with AT1R and the inhibitory effect of ATR2 on AT1R. These data suggest that AT2R inhibits ligand-induced AT1R signaling through the PKC-dependent pathway.
Asunto(s)
Membrana Celular/metabolismo , Proteína Quinasa C/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal/fisiología , Membrana Celular/genética , Endosomas/genética , Endosomas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Proteína Quinasa C/genética , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 2/genéticaRESUMEN
Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. Tcf8-heterozygous knockout mice are more permissive than wild-type mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMP1 overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer.
Asunto(s)
Proteínas de Homeodominio/fisiología , Melanoma Experimental/irrigación sanguínea , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Factores de Transcripción/fisiología , Animales , Cromatina/genética , Dipéptidos/farmacología , Endotelio Vascular/fisiología , Endotelio Vascular/fisiopatología , Proteínas de Homeodominio/genética , Homeostasis , Humanos , Melanoma Experimental/patología , Metaloproteasas/antagonistas & inhibidores , Ratones , Ratones Noqueados , Invasividad Neoplásica , Neovascularización Patológica/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Venas Umbilicales , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
We have recently reported that transcription factor 8 (TCF8) negatively regulates pathological angiogenesis by regulating endothelial invasiveness by acting as a transcriptional attenuator of matrix metalloproteinase 1. TCF8 also modulates cell-matrix and cell-cell adhesion; however molecular mechanism of this TCF8 function remains obscure. Here, we provide evidence that TCF8 activates R-Ras, another class of angiogenic regulator, to suppress angiogenesis by a mechanism other than a transcriptional attenuator. Tube formation by human umbilical vein endothelial cells (HUVECs) facilitated by TCF8 suppression was significantly inhibited by the expression of constitutive active mutant of R-Ras. When we examined the mRNA expression levels of R-Ras regulators, no significant changes were observed to explain the R-Ras activation by TCF8. Interestingly, we found that TCF8 bound to CalDAG-GEFIII, an R-Ras activator, in the cytosol, indicating that TCF8 emanates signaling for R-Ras activation from cytosol to regulate angiogenesis negatively.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Neovascularización Fisiológica , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Homeodominio/genética , Humanos , Mutación , Neovascularización Fisiológica/genética , Transducción de Señal , Factores de Transcripción/genética , Venas Umbilicales/citología , Venas Umbilicales/enzimología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Factores de Intercambio de Guanina Nucleótido ras , Proteínas ras/genéticaRESUMEN
Host defense consists of two main aspects, namely, immune response to invading pathogens and suppression of tumor development. A family of transcription factors, IFN regulatory factors (IRFs), has recently gained much attention in terms of its critical role in linking these two aspects of host defense, wherein IRF5 was previously shown to play a critical role in the induction of proinflammatory cytokines by activation of Toll-like receptors. In the present study, using IRF5 gene-targeted mice (Irf5(-/-) mice), we demonstrate another facet of the IRF5 function in the regulation of immune response and tumor suppression. We show that IRF5 is critical for antiviral immunity by showing that Irf5(-/-) mice are highly vulnerable to viral infections, accompanied by a decrease in type I IFN induction in the sera. Furthermore, we show that Irf5(-/-) fibroblasts are resistant to apoptosis upon viral infection, resulting in an enhanced viral propagation. Finally, we provide evidence that IRF5 is critical for the induction of apoptosis, but not in cell cycle arrest, in response to DNA damage and that IRF5 functions as a tumor suppressor by acting on a pathway that may be distinct from that for p53. These results, together with the dual regulation of IRF5 gene expression by IFN signaling and p53, may provide a new link in the transcriptional network underlying antiviral immunity and tumor suppression.
Asunto(s)
Apoptosis/inmunología , Regulación de la Expresión Génica/inmunología , Factores Reguladores del Interferón/inmunología , Virosis/inmunología , Animales , Ciclo Celular/inmunología , Cartilla de ADN , Interleucina-6/sangre , Macrófagos/inmunología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/inmunología , Virosis/genéticaRESUMEN
The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.