RESUMO
Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.
Assuntos
Matriz Extracelular/fisiologia , Prosencéfalo/irrigação sanguínea , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologia , Animais , Proteína Axina/fisiologia , Barreira Hematoencefálica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Remodelação Vascular , Peixe-ZebraRESUMO
The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate "Ripoptosome" is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIP(L) prevents Ripoptosome formation, whereas, intriguingly, cFLIP(S) promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the "rheostat" that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.
Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Caspase 8/fisiologia , Proteínas Inibidoras de Apoptose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Isoformas de Proteínas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gene expression profiling of various cell lineages has provided invaluable insights into the molecular mechanisms regulating cellular development and differentiation. However, in vivo molecular profiling of rare and interspersed cell populations, such as endothelial cells, has remained challenging. We have generated a versatile floxed translating ribosome affinity purification (TRAP) mouse model, mCherryTRAP, for Cre-dependent translational profiling of distinct cell lineages from intact tissues. To identify cell type-specific transcripts using TRAP, the data have to be filtered to remove both background transcripts not expressed in the profiled cell type and transcripts expressed in all cell populations of the tissue/organ. Filtering has previously been achieved using transcribed RNA from the tissue/organ. Using the mCherryTRAP model, we demonstrate extensive differential expression of RNAs between the translatome and transcriptome of embryonic brains and kidneys. We evaluate the implications of these data for TRAP studies of abundant and rare cell populations. Finally, we demonstrate the applicability of the technology to study organ-specific endothelial cell differentiation.
Assuntos
Camundongos/genética , Modelos Animais , Biossíntese de Proteínas , Análise de Sequência de RNA , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Perfilação da Expressão Gênica , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/análise , Proteína Vermelha FluorescenteRESUMO
Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIß activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca(2+) sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions.
Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Calmodulina/metabolismo , Membranas Intracelulares/metabolismo , Chaperonas Moleculares/metabolismo , Rede trans-Golgi/metabolismo , Animais , ATPases Transportadoras de Arsenito/genética , Células COS , Cálcio/metabolismo , Calmodulina/genética , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Chaperonas Moleculares/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Rede trans-Golgi/genéticaRESUMO
Primary endothelial cells are fully resistant to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Here, we demonstrate that certain environmental conditions, such as exposure to the widespread allergen nickel, can dramatically increase the susceptibility of naturally resistant primary endothelial cells or keratinocytes to TRAIL-induced apoptosis. While nickel treatment increased surface expression of the apoptosis-inducing TRAIL receptors TRAIL-R1 and TRAIL-R2, it also up-regulated the apoptosis-deficient TRAIL-R4, suggesting that modulation of TRAIL receptor expression alone is unlikely to fully account for the dramatic sensitization effect of nickel. Further analysis of candidate mediators revealed that nickel strongly repressed c-FLIP at mRNA and protein levels. Accordingly, increased activation of Caspase-8 and Caspase-3 following nickel treatment was observed. Importantly, depletion of c-FLIP by RNA interference could largely recapitulate the effect of nickel and sensitize endothelial cells to TRAIL-dependent apoptosis in the absence of nickel pre-treatment. Conversely, ectopic expression of c-FLIP(L) largely protected nickel-treated cells from TRAIL-mediated apoptosis. Our data demonstrate that one key mechanism of sensitization of primary human endothelial cells or keratinocytes is transcriptional down-regulation of c-FLIP. We hypothesize that environmental factors, exemplified by the contact allergen nickel, strongly modulate death ligand sensitivity of endothelial cells and keratinocytes thus influencing vascular and epidermal function and integrity under physiological and pathophysiological conditions.
Assuntos
Alérgenos/imunologia , Apoptose/efeitos dos fármacos , Células Endoteliais/citologia , Queratinócitos/citologia , Níquel/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Alérgenos/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Níquel/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Transcrição Gênica/efeitos dos fármacos , Veias Umbilicais/citologiaRESUMO
The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system, and it is formed by highly specialized endothelial cells. To explore the molecular mechanisms defining the unique nature of vascular development and differentiation in the brain, we generated high-resolution gene expression profiles of mouse embryonic brain endothelial cells using translating ribosome affinity purification and single-cell RNA sequencing. We compared the brain vascular translatome with the vascular translatomes of other organs and analyzed the vascular translatomes of the brain at different time points during embryonic development. Because canonical Wnt signaling is implicated in the formation of the blood-brain barrier, we also compared the brain endothelial translatome of wild-type mice with that of mice lacking the transcriptional cofactor ß-catenin (Ctnnb1). Our analysis revealed extensive molecular changes during the embryonic development of the brain endothelium. We identified genes encoding brain endothelium-specific transcription factors (Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, and Zic3) that are associated with maturation of the blood-brain barrier and act downstream of the Wnt-ß-catenin signaling pathway. Profiling of individual brain endothelial cells revealed substantial heterogeneity in the population. Nevertheless, the high abundance of Foxf2, Foxq1, Ppard, or Zic3 transcripts correlated with the increased expression of genes encoding markers of brain endothelial cell differentiation. Expression of Foxf2 and Zic3 in human umbilical vein endothelial cells induced the production of blood-brain barrier differentiation markers. This comprehensive data set may help to improve the engineering of in vitro blood-brain barrier models.
Assuntos
Encéfalo/embriologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Encéfalo/citologia , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Camundongos , Camundongos TransgênicosRESUMO
FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Queratinócitos/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/deficiência , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Homeostase , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/patologiaRESUMO
A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP(L) and not cFLIP(S) interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.
Assuntos
Apoptose , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptor fas/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína Ligante Fas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-kappaB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-kappaB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-kappaB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-kappaB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-kappaB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-kappaB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.
Assuntos
Caspase 8/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratinócitos/citologia , Mutagênese , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/fisiologia , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Axonemal dyneins are large motor protein complexes generating the force for the movement of eukaryotic cilia and flagella. Disruption of axonemal dynein function leads to loss of ciliary motility and can result in male infertility or lateralization defects. Here, we report the molecular analysis of a murine gene encoding the dynein axonemal light intermediate chain Dnali1. The Dnali1 gene is localized on chromosome 4 and consists of six exons. It is predominantly expressed within the testis but at a lower level Dnali1 transcripts were also observed in different murine tissues, which exhibit cilia. Two transcript variants were detected, generated by the usage of two alternative polyadenylation signals within exon 6. Antibodies were raised against a GST-Dnali1 fusion protein and used to localize Dnali1 within differentiating male germ cells. Dnali1 is strongly expressed in spermatids but was also detected in spermatocytes. Moreover, the Dnali1 protein was localized in cilia of the trachea as well as in flagella of mature sperm supporting its function as an axonemal dynein. To identify putative Dnali1 interacting polypeptides, a yeast two-hybrid approach was performed using a murine testicular cDNA library. By this assay, the C-terminal part of the cytoplasmic dynein heavy chain 1 was identified as a putative interacting polypeptide of Dnali1. The interaction between the axonemal and the cytoplasmic dynein fragments was proven by co-immuno and co-localization experiments.