RESUMO
Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas , Janus Quinase 2 , Transtornos Mieloproliferativos , Transgenes , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Janus Quinase 2/biossíntese , Janus Quinase 2/genética , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologiaRESUMO
Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Humanos , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/toxicidade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAPRESUMO
A large body of data demonstrates that interferon regulatory factor 5 (IRF5) and nuclear factor kappa B (NF-κB) are the two major transcription factors in classically activated macrophages responsible for the transcriptional control of proinflammatory genes. Although recent evidence suggests that IRF5 interacts with certain members of the nuclear factor kappa B pathway, the extent of cooperation and its implications in disease are ambiguous. Since both pathways are known for their strong contributions in TLR8 signaling we used the human monocytic cell line THP-1.Dual, featuring gene reporters for NF-κB and IRFs, to simultaneously study the roles of IRF5 and the NF-κB subunit p65 in TLR8-mediated gene reporter activities. Furthermore, we profiled from these cells the proinflammatory cytokines involved in the differentiation of TH1 and TH17 cells. After ablation of IRF5 and/or p65 we activated the resultant cells with the TLR8 agonists R848 or the psoriasis-associated antimicrobial peptide LL-37 complexed with ssRNA and demonstrate that IRF5 deficiency drastically impairs the secretion of IL-1ß, IL-6, IL-12, IL-23 and TNFα. In contrast, the lack of p65 impaired only IL-6, IL-12, and IL-23 secretion. Furthermore, we discovered that upon TLR8 stimulation, IRF5 but not NF-κB signaling is essential to provide a cytokine milieu supporting TH1 responses. Additionally, we demonstrate that IRF5 and NF-κB cooperate to provide a cytokine milieu supporting TH17 responses. Therefore, the distinct role of IRF5 in the intricate signaling network downstream of TLR8 may open new treatment options interfering with but not disrupting NF-κB signaling in human diseases.
Assuntos
Inflamação/metabolismo , Fatores Reguladores de Interferon/metabolismo , NF-kappa B/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Citocinas/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Humanos , Imidazóis/farmacologia , Fatores Reguladores de Interferon/genética , NF-kappa B/genética , Elementos de Resposta , Transdução de Sinais , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo , CatelicidinasRESUMO
The balance between stem cell quiescence and proliferation in skeletal muscle is tightly controlled, but perturbed in a variety of disease states. Despite progress in identifying activators of stem cell proliferation, the niche factor(s) responsible for quiescence induction remain unclear. Here we report an in vivo imaging-based screen which identifies Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, as a potent inducer of muscle stem cell (MuSC, satellite cell) quiescence. OSM is produced by muscle fibers, induces reversible MuSC cell cycle exit, and maintains stem cell regenerative capacity as judged by serial transplantation. Conditional OSM receptor deletion in satellite cells leads to stem cell depletion and impaired regeneration following injury. These results identify Oncostatin M as a secreted niche factor responsible for quiescence induction, and for the first time establish a direct connection between induction of quiescence, stemness, and transplantation potential in solid organ stem cells.
Assuntos
Músculo Esquelético/metabolismo , Oncostatina M/fisiologia , Células-Tronco/citologia , Alelos , Animais , Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Proliferação de Células , Feminino , Humanos , Interleucina-6/metabolismo , Luminescência , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.
Assuntos
Perfilação da Expressão Gênica , Fígado/parasitologia , Macaca mulatta/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/genética , Animais , Feminino , MasculinoRESUMO
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
Assuntos
Fator D do Complemento/antagonistas & inibidores , Via Alternativa do Complemento/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fator D do Complemento/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Hearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss. RESULTS: In order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach. CONCLUSION: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.
Assuntos
Transdiferenciação Celular , Células Ciliadas Auditivas Internas/citologia , MicroRNAs/metabolismo , Órgão Espiral/citologia , Regeneração , Animais , Linhagem Celular , Técnicas de Introdução de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/genética , Análise de Sequência de RNARESUMO
LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/ß-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/ß-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/ß-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/ß-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/ß-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.
Assuntos
Fígado/citologia , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Proliferação de Células , Citocromo P-450 CYP2E1/metabolismo , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/metabolismo , Homeostase , Antígeno Ki-67/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Regeneração Hepática , Tamanho do Órgão , Transdução de Sinais , beta-Galactosidase/metabolismoRESUMO
Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Endoteliais/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , Hipertensão Pulmonar/patologia , Artéria Pulmonar/patologia , Envelhecimento/patologia , Animais , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Densitometria , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Monocrotalina , Fosforilação/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Sprague-Dawley , Sístole/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.
Assuntos
Linfócitos B Reguladores/imunologia , Caspases/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B Reguladores/patologia , Caspases/genética , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4-agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.
Assuntos
Densidade Óssea , Osso e Ossos/metabolismo , Glicoproteínas/sangue , Receptores de LDL/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Agrina/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Linhagem Celular , Feminino , Colo do Fêmur/microbiologia , Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Relacionadas a Receptor de LDL , Masculino , Camundongos Knockout , Microscopia Confocal , Junção Neuromuscular/metabolismo , Osteoblastos/metabolismo , Osteócitos/metabolismo , Osteogênese/genética , Ligação Proteica , Ratos Wistar , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microtomografia por Raio-XRESUMO
Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.
Assuntos
Intestinos/embriologia , Rim/embriologia , Receptores Acoplados a Proteínas G/fisiologia , Pele/embriologia , Via de Sinalização Wnt/fisiologia , Animais , Southern Blotting , Primers do DNA/genética , Componentes do Gene , Genótipo , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologia , Via de Sinalização Wnt/genéticaRESUMO
Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.
Assuntos
Alelos , Cardiopatias Congênitas/genética , Oxirredutases N-Desmetilantes/genética , Animais , Caderinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Comunicação Interventricular/genética , Comunicação Interventricular/metabolismo , Comunicação Interventricular/patologia , Histona Desmetilases , Homozigoto , Camundongos , Camundongos Knockout , Oxirredutases N-Desmetilantes/metabolismo , Fosforilação , Mutação Puntual , Gravidez , Ligação ProteicaRESUMO
BACKGROUND: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.
Assuntos
Aldeído Liases/deficiência , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/prevenção & controle , Aldeído Liases/metabolismo , Animais , Encéfalo/metabolismo , Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/complicações , Fatores de Transcrição Forkhead/metabolismo , Hipersensibilidade Tardia/sangue , Hipersensibilidade Tardia/complicações , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Memória Imunológica/imunologia , Integrases/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Ovinos , Esfingolipídeos/metabolismo , Baço/imunologia , Baço/patologia , Análise de Sobrevida , Timo/imunologia , Timo/patologiaRESUMO
Pulmonary surfactant levels within the alveoli are tightly regulated to maintain lung volumes and promote efficient gas exchange across the air/blood barrier. Quantitative and qualitative abnormalities in surfactant are associated with severe lung diseases in children and adults. Although the cellular and molecular mechanisms that control surfactant metabolism have been studied intensively, the critical molecular pathways that sense and regulate endogenous surfactant levels within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. In this study, we demonstrate that expression of an orphan G protein-coupled receptor, GPR116, in the murine lung is developmentally regulated, reaching maximal levels 1 day after birth, and is highly expressed on the apical surface of alveolar type I and type II epithelial cells. To define the physiological role of GPR116 in vivo, mice with a targeted mutation of the Gpr116 locus, Gpr116(Δexon17), were generated. Gpr116(Δexon17) mice developed a profound accumulation of alveolar surfactant phospholipids at 4 weeks of age (12-fold) that was further increased at 20 weeks of age (30-fold). Surfactant accumulation in Gpr116(Δexon17) mice was associated with increased saturated phosphatidylcholine synthesis at 4 weeks and the presence of enlarged, lipid-laden macrophages, neutrophilia, and alveolar destruction at 20 weeks. mRNA microarray analyses indicated that P2RY2, a purinergic receptor known to mediate surfactant secretion, was induced in Gpr116(Δexon17) type II cells. Collectively, these data support the concept that GPR116 functions as a molecular sensor of alveolar surfactant lipid pool sizes by regulating surfactant secretion.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Macrófagos/metabolismo , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Receptores Acoplados a Proteínas G/genética , Mucosa Respiratória/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Células Epiteliais/patologia , Éxons , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fosfatidilcolinas/biossíntese , Alvéolos Pulmonares/patologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Mucosa Respiratória/patologia , Transdução de SinaisRESUMO
During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Animais , Hemorreologia , Homeostase , Camundongos , Receptores de Lisoesfingolipídeo/sangueRESUMO
The rod-derived cone viability factors, RdCVF and RdCVF2, have potential therapeutical interests for the treatment of inherited photoreceptor degenerations. In the mouse lacking Nxnl2, the gene encoding RdCVF2, the progressive decline of the visual performance of the cones in parallel with their degeneration, arises due to the loss of trophic support from RdCVF2. In contrary, the progressive loss of rod visual function of the Nxnl2-/- mouse results from a decrease in outer segment length, mediated by a cell autonomous mechanism involving the putative thioredoxin protein RdCVF2L, the second spliced product of the Nxnl2 gene. This novel signaling mechanism extends to olfaction as shown by the progressive impairment of olfaction in aged Nxnl2-/- mice and the protection of olfactory neurons by RdCVF2. This study shows that Nxnl2 is a bi-functional gene involved in the maintenance of both the function and the viability of sensory neurons.
Assuntos
Sobrevivência Celular/genética , Proteínas do Olho/genética , Splicing de RNA , Células Receptoras Sensoriais/citologia , Tiorredoxinas/genética , Animais , Células Cultivadas , Proteínas do Olho/metabolismo , Camundongos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Receptoras Sensoriais/metabolismo , Tiorredoxinas/metabolismoRESUMO
Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.