RESUMO
Delta-like 4 (DLL4), a membrane-bound ligand belonging to the Notch signaling family, plays a fundamental role in vascular development and angiogenesis. We identified a conserved microRNA family, miR-30, which targets DLL4. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR-30 mimics into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3'UTR up-regulated dll4 and synergized with Vegfa signaling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during Kaposi sarcoma herpesvirus (KSHV) infection attenuated viral induction of DLL4. Together these results demonstrate that the highly conserved molecular targeting of DLL4 by the miR-30 family regulates angiogenesis.
Assuntos
Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Neovascularização Fisiológica , Animais , Sequência de Bases , Linhagem Celular , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Regulação da Expressão Gênica no Desenvolvimento , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologiaRESUMO
Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Endotélio Vascular/fisiologia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sistema Linfático/fisiologia , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Sarcoma de Kaposi/virologia , Proteínas Adaptadoras de Transdução de Sinal , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Proteína Jagged-1 , Sistema Linfático/citologia , Sistema Linfático/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/genética , Receptor Notch4 , Receptores Notch/genética , Sarcoma de Kaposi/genética , Proteínas Serrate-Jagged , Transdução de Sinais , Regulação para CimaRESUMO
The involvement of Toll-like receptor 4 (TLR4) in immunity against human herpesviruses has not been previously demonstrated. We show that infection of endothelial cells with Kaposi sarcoma herpesvirus (KSHV), a human oncogenic virus, leads to rapid suppression of TLR4 expression. This is a mechanism of immune escape as TLR4 mediates innate immunity against KSHV. In vitro, cells lacking TLR4 are more susceptible to KSHV infection, whereas activation of TLR4 protects cells from infection. In vivo, HIV-1-infected individuals carrying a mutant TLR4 allele appear more likely to have multicentric Castleman's disease, a lymphoproliferation associated with enhanced KSHV replication. ERK activation by KSHV structural proteins and the KSHV-encoded vGPCR plays a key role in the TLR4 downregulation, whereas the KSHV vIRF1 also contributes to this effect. Our findings reveal a role for TLR4 in innate immunity against herpesviruses and suggest the potential use of TLR4 agonists for the treatment of KSHV-related neoplasms.
Assuntos
Herpesvirus Humano 8/imunologia , Sarcoma de Kaposi/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Cultivadas , Regulação para Baixo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Expressão Gênica , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Herpesvirus Humano 8/fisiologia , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The protein kinase B-RAF is mutated in approximately 8% of human cancers. Here we show that presumptive mutants of the closely related kinase, C-RAF, were detected in only 4 of 545 (0.7%) cancer cell lines. The activity of two of the mutated proteins is not significantly different from that of wild-type C-RAF and these variants may represent rare human polymorphisms. The basal and B-RAF-stimulated kinase activities of a third variant are unaltered but its activation by RAS is significantly reduced, suggesting that it may act in a dominant-negative manner to modulate pathway signaling. The fourth variant has elevated basal kinase activity and is hypersensitive to activation by RAS but does not transform mammalian cells. Furthermore, when we introduce the equivalent of the most common cancer mutation in B-RAF (V600E) into C-RAF, it only has a weak effect on kinase activity and does not convert C-RAF into an oncogene. This lack of activation occurs because C-RAF lacks a constitutive charge within a motif in the kinase domain called the N-region. This fundamental difference in RAF isoform regulation explains why B-RAF is frequently mutated in cancer whereas C-RAF mutations are rare.