RESUMO
Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.
Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Sequência de Aminoácidos/genética , Animais , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Morte Celular/efeitos dos fármacos , Simulação por Computador , Bases de Dados Genéticas , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neuropeptídeos/metabolismo , Filogenia , Transdução de Sinais/fisiologiaRESUMO
Producing the neuronal diversity required to adequately discriminate all elements of somatosensation is a complex task during organogenesis. The mechanisms guiding this process during dorsal root ganglion (DRG) sensory neuron specification remain poorly understood. Here, we show that the p75 neurotrophin receptor interacts with Ret and its GFRα co-receptor upon stimulation with glial cell line-derived neurotrophic factor (GDNF). Furthermore, we demonstrate that p75 is required for GDNF-mediated Ret activation, survival, and cell surface localization of Ret in DRG neurons. In mice in which p75 is deleted specifically within sensory neurons beginning at E12.5, we observe that approximately 20% of neurons are lost between P14 and adulthood, and these losses selectively occur within a subpopulation of Ret+ nonpeptidergic nociceptors, with neurons expressing low levels of Ret impacted most heavily. These results suggest that p75 is required for the development of the nonpeptidergic nociceptor lineage by fine-tuning Ret-mediated trophic support.
Assuntos
Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Integrases/metabolismo , Ligantes , Camundongos , Nociceptividade/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Peptídeos/metabolismoRESUMO
Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from early to late endosome for degradation. Our studies provide insight into mechanisms by which NUMB and NUMBL promote cardiomyocyte cell cycle withdrawal and highlight previously unsuspected connections between pathways that are important for cardiomyocyte cell cycle reentry, with relevance to ventricular noncompaction cardiomyopathy and regenerative medicine.
Assuntos
Ciclo Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Proteínas de Ciclo Celular , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor ErbB-2/genética , Transdução de Sinais/genética , Fatores de Transcrição , Proteínas de Sinalização YAP , Proteínas rab de Ligação ao GTP , proteínas de unión al GTP Rab7RESUMO
Several lines of evidence suggest that neuregulin 1 (NRG1) signaling may influence cognitive function and neuropathology in Alzheimer's disease (AD). To test this possibility, full-length type I or type III NRG1 was overexpressed via lentiviral vectors in the hippocampus of line 41 AD mouse. Both type I and type III NRG1 improves deficits in the Morris water-maze behavioral task. Neuropathology was also significantly ameliorated. Decreased expression of the neuronal marker MAP2 and synaptic markers PSD95 and synaptophysin in AD mice was significantly reversed. Levels of Aß peptides and plaques were markedly reduced. Furthermore, we showed that soluble ectodomains of both type I and type III NRG1 significantly increased expression of Aß-degrading enzyme neprilysin (NEP) in primary neuronal cultures. Consistent with this finding, immunoreactivity of NEP was increased in the hippocampus of AD mice. These results suggest that NRG1 provides beneficial effects in candidate neuropathologic substrates of AD and, therefore, is a potential target for the treatment of AD.
Assuntos
Doença de Alzheimer/terapia , Neuregulina-1/fisiologia , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Cognição , Feminino , Hipocampo/patologia , Humanos , Aprendizagem em Labirinto , Camundongos Transgênicos , Neprilisina/metabolismo , RatosRESUMO
Composite colloidal structures with multi-functional properties have wide applications in targeted delivery of therapeutics and imaging contrast molecules and high-throughput molecular bio-sensing. We have constructed a multifunctional composite magnetic nanobowl using the bottom-up approach on an asymmetric silica/polystyrene Janus template consisting of a silica shell around a partially exposed polystyrene core. The nanobowl consists of a silica bowl and a gold exterior shell with iron oxide magnetic nanoparticles sandwiched between the silica and gold shells. The nanobowls were characterized by electron microscopy, atomic force microscopy, magnetometry, vis-NIR and FTIR spectroscopy. Magnetically vectored transport of these nanobowls was ascertained by time-lapsed imaging of their flow in fluid through a porous hydrogel under a defined magnetic field. These magnetically-responsive nanobowls show distinct surface enhanced Raman spectroscopy (SERS) imaging capability. The PEGylated magnetically-responsive nanobowls show size-dependent cellular uptake in vitro.
Assuntos
Ouro/química , Nanopartículas/química , Dióxido de Silício/química , Análise Espectral Raman , Linhagem Celular Tumoral , Humanos , PoliestirenosRESUMO
The p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily, is required as a co-receptor for the Nogo receptor (NgR) to mediate the activity of myelin-associated inhibitors such as Nogo, MAG, and OMgp. p45/NRH2/PLAIDD is a p75 homologue and contains a death domain (DD). Here we report that p45 markedly interferes with the function of p75 as a co-receptor for NgR. P45 forms heterodimers with p75 and thereby blocks RhoA activation and inhibition of neurite outgrowth induced by myelin-associated inhibitors. p45 binds p75 through both its transmembrane (TM) domain and DD. To understand the underlying mechanisms, we have determined the three-dimensional NMR solution structure of the intracellular domain of p45 and characterized its interaction with p75. We have identified the residues involved in such interaction by NMR and co-immunoprecipitation. The DD of p45 binds the DD of p75 by electrostatic interactions. In addition, previous reports suggested that Cys257 in the p75 TM domain is required for signaling. We found that the interaction of the cysteine 58 of p45 with the cysteine 257 of p75 within the TM domain is necessary for p45-p75 heterodimerization. These results suggest a mechanism involving both the TM domain and the DD of p45 to regulate p75-mediated signaling.
Assuntos
Multimerização Proteica , Receptor de Fator de Crescimento Neural/química , Receptor de Fator de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Receptores de Superfície Celular/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Soluções , Relação Estrutura-Atividade , Regulação para CimaRESUMO
Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.
Assuntos
Aborto Espontâneo/genética , Receptores ErbB/genética , Fertilidade/genética , Complicações na Gravidez/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/genética , Decídua/metabolismo , Endometriose/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Gravidez , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Transdução de Sinais/genética , Proteína Quinase 1 Deficiente de Lisina WNK , Proteína Wnt4/genéticaRESUMO
Fas-associated death domain (DD) adaptor (FADD), a member of the DD superfamily, contains both a DD and a death effector domain (DED) that are important in mediating FAS ligand-induced apoptotic signaling. P45 is a unique member of the DD superfamily in that it has a domain with sequence and structural characteristics of both DD and DED. We show that p45 forms a complex with FADD and diminishes Fas-FADD mediated death signaling. The DED of FADD is required for the complex formation with p45. Following spinal cord injury, transgenic mice over-expressing p45 exhibit increased neuronal survival, decreased retraction of corticospinal tract fibers and improved functional recovery. Understanding p45-mediated cellular and molecular mechanisms may provide insights into facilitating nerve regeneration in humans.
Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Receptores de Morte Celular/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Morte Celular , Sobrevivência Celular , Ativação Enzimática , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/química , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , Transdução de Sinais , Antígenos Thy-1/metabolismoRESUMO
BACKGROUND: ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). METHODS: To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. RESULTS: Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. CONCLUSIONS: These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.
Assuntos
Medula Óssea/virologia , Proteínas de Ligação a DNA/metabolismo , Leucemia Experimental/virologia , Vírus da Leucemia Murina de Moloney/fisiologia , Proteínas Nucleares/metabolismo , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/virologia , Replicação Viral , Animais , Proteínas de Ligação a DNA/deficiência , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiênciaRESUMO
NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.
Assuntos
Proteínas de Transporte/metabolismo , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Estresse Oxidativo , Peroxidases/metabolismo , Deficiências na Proteostase/enzimologia , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cisteína , Dano ao DNA , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fibroblastos/enzimologia , Fibroblastos/patologia , Glutationa Peroxidase , Proteínas de Choque Térmico/genética , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Peroxidases/genética , Ligação Proteica , Dobramento de Proteína , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , TransfecçãoRESUMO
Reverse signaling by ephrin-As upon binding EphAs controls axon guidance and mapping. Ephrin-As are GPI-anchored to the membrane, requiring that they complex with transmembrane proteins that transduce their signals. We show that the p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75(NTR) and ephrin-A colocalize within caveolae along retinal axons and form a complex required for Fyn phosphorylation upon binding EphAs, activating a signaling pathway leading to cytoskeletal changes. In vitro, retinal axon repulsion to EphAs by ephrin-A reverse signaling requires p75(NTR), but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75(NTR) knockout mice have aberrant anterior shifts in retinal axon terminations in superior colliculus, consistent with diminished repellent activity mediated by graded ephrin-A reverse signaling induced by graded collicular EphAs. We conclude that p75(NTR) is a signaling partner for ephrin-As and the ephrin-A- p75(NTR) complex reverse signals to mediate axon repulsion required for guidance and mapping.
Assuntos
Axônios/fisiologia , Mapeamento Encefálico , Efrinas/metabolismo , Células Ganglionares da Retina/citologia , Transdução de Sinais/fisiologia , Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Receptores de Fator de Crescimento Neural , Retina/citologia , Células Ganglionares da Retina/fisiologia , Vias Visuais/fisiologiaRESUMO
erbB2/Her2, a ligandless receptor kinase, has pleiotropic effects on mammalian development and human disease. The absence of erbB2 signaling in cardiac myocytes results in dilated cardiomyopathy in mice, resembling the cardiotoxic effects observed in a subset of breast cancer patients treated with the anti-Her2 antibody herceptin. Emerging evidence suggests that erbB2 is pivotal for integrating signaling networks involving multiple classes of extracellular signals. However, its role in G protein-coupled receptor (GPCR) signaling remains undefined. Because the activation of the MAPK pathway through GPCR signaling is important for cardiac homeostasis, we investigated whether erbB2 is required for GPCR-mediated MAPK signaling in wild-type and heart-specific erbB2 mutant mice. Here we demonstrate that erbB2, but not EGF receptor, is essential for MAPK activation induced by multiple GPCR agonists in cardiac myocytes. erbB2 is immunocomplexed with a GPCR in vivo and is transactivated after ligand treatment in vitro. Coexpression of erbB2 with GPCRs in heterologous cells results in ligand-dependent complex formation and MAPK activation. Furthermore, MAPK activation and cardiac contractility are markedly impaired in heart-specific erbB2 mutants infused with a GPCR agonist. These results reveal an essential mechanism requiring erbB2 as a coreceptor for GPCR signaling in the heart. The obligatory role of erbB2 in GPCR-dependent signaling may also be important in other cellular systems.
Assuntos
Miocárdio/metabolismo , Receptor ErbB-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Chlorocebus aethiops , Ativação Enzimática , Ligantes , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica , Receptor ErbB-2/genéticaRESUMO
ErbB2 protein is essential for the development of Schwann cells and for the normal fiber growth and myelin formation of peripheral nerves. We have investigated the fate of the otocyst-derived inner ear sensory neurons in the absence of ErbB2 using ErbB2 null mutants. Afferent innervation of the ear sensory epithelia shows numerous fibers overshooting the organ of Corti, followed by a reduction of those fibers in near term embryos. This suggests that mature Schwann cells do not play a role in targeting or maintaining the inner ear innervation. Comparable to the overshooting of nerve fibers, sensory neurons migrate beyond their normal locations into unusual positions in the modiolus. They may miss a stop signal provided by the Schwann cells that are absent as revealed with detailed histology. Reduction of overshooting afferents may be enhanced by a reduction of the neurotrophin Ntf3 transcript to about 25% of wild type. Ntf3 transcript reductions are comparable to an adult model that uses a dominant negative form of ErbB4 expressed in the supporting cells and Schwann cells of the organ of Corti. ErbB2 null mice retain afferents to inner hair cells possibly because of the prominent expression of the neurotrophin Bdnf in developing hair cells. Despite the normal presence of Bdnf transcript, afferent fibers are disoriented near the organ of Corti. Efferent fibers do not form an intraganglionic spiral bundle in the absence of spiral ganglia and appear reduced and disorganized. This suggests that either ErbB2 mediated alterations in sensory neurons or the absence of Schwann cells affects efferent fiber growth to the organ of Corti.
Assuntos
Orelha Interna/inervação , Orelha Interna/fisiologia , Receptor ErbB-2/deficiência , Animais , Padronização Corporal , Nervo Coclear/citologia , Nervo Coclear/embriologia , Humanos , Camundongos , Camundongos Knockout , Neurônios Aferentes/fisiologia , Receptor ErbB-2/genéticaRESUMO
The ERBB2/HER2/NEU receptor tyrosine kinase gene is amplified in up to 30% of human breast cancers. The frequent and specific selection of this receptor kinase gene for amplification in breast cancer implies that it has important normal functions in the mammary gland. To investigate the functions of ErbB2 during normal mouse mammary gland development, we transplanted mammary buds from genetically rescued ErbB2(-/-) embryos that express ErbB2 in the cardiac muscle. ErbB2(-/-) mammary buds transplanted to a wild-type mammary fat pad support outgrowth of an epithelial tree that advances only slowly through the mammary fat pad at puberty. This penetration defect is associated with structural defects in terminal end buds, characterized by a decrease in body cell number, an increased presence of cap-like cells in the prelumenal compartment, and the presence of large luminal spaces. Lobuloalveolar development was not affected in glands that developed from ErbB2(-/-) transplanted tissue. The results may have implications for the aggressive phenotypes associated with ERBB2-overexpressing mammary carcinomas.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptor ErbB-2/fisiologia , Animais , Epitélio/anormalidades , Epitélio/crescimento & desenvolvimento , Feminino , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Animais/transplante , Neoplasias Mamárias Animais/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ratos , Receptor ErbB-2/deficiência , Receptor ErbB-2/genéticaRESUMO
Approximately 70% of human breast cancers are estrogen receptor alpha (ERalpha)-positive, but the origins of ERalpha-positive and -negative tumors remain unclear. Hormonal regulation of mammary gland development in mice is similar to that in humans; however, most mouse models produce only ERalpha-negative tumors. In addition, these mouse tumors metastasize at a low rate relative to human breast tumors. We report here that somatic mutations of p53 in mouse mammary epithelial cells using the Cre/loxP system leads to ERalpha-positive and -negative tumors. p53 inactivation under a constitutive active WAPCre(c) in prepubertal/pubertal mice, but not under MMTVCre in adult mice, leads to the development of ERalpha-positive tumors, suggesting that target cells or developmental stages can determine ERalpha status in mammary tumors. Importantly, these tumors have a high rate of metastasis. An inverse relationship between the number of targeted cells and median tumor latency was also observed. Median tumor latency reaches a plateau when targeted cell numbers exceed 20%, implying the existence of saturation kinetics for breast carcinogenesis. Genetic alterations commonly observed in human breast cancer including c-myc amplification and Her2/Neu/erbB2 activation were seen in these mouse tumors. Thus, this tumor system reproduces many important features of human breast cancer and provides tools for the study of the origins of ERalpha-positive and -negative breast tumors in mice.
Assuntos
Genes p53/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Mutação , Receptores de Estrogênio/biossíntese , Alelos , Animais , Modelos Animais de Doenças , Receptor alfa de Estrogênio , Feminino , Inativação Gênica , Integrases/genética , Neoplasias Mamárias Experimentais/metabolismo , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Metástase Neoplásica , Proteínas Virais/genéticaRESUMO
Corticotropin-releasing factor (CRF) and its paralogues urocortin (Ucn)I, -II, and -III signal by activating their receptors, CRF receptors (CRFR)1 and -2, to maintain homeostasis through endocrine, autonomic, and behavioral responses. CRFR2 is found in cardiomyocytes and in endothelial and smooth muscle cells of the systemic vasculature. Echocardiography and cardiac catheterization were used in mice to assess the physiologic effects of i.v. UcnII and CRFR2 deficiency on left ventricular function and the systemic vasculature. UcnII treatment augmented heart rate, exhibited potent inotropic and lusitropic actions on the left ventricle, and induced a downward shift of the diastolic pressure-volume relation. UcnII also reduced systemic arterial pressure, associated with a lowering of systemic arterial elastance (end-systolic pressure/stroke volume) and systemic vascular resistance. CRFR2-deficient mice showed no alteration in cardiac contractility or blood pressure in response to UcnII administration, suggesting that the effects of UcnII are specific to CRFR2 function. Pretreatment with a beta-adrenergic receptor antagonist, esmalol, had no effect on the inotropic or lusitropic effects of UcnII in vivo, indicating that its actions are independent of beta-adrenergic receptors. Single i.v. bolus administration of UcnII to a heart failure model (muscle-specific LIM protein-deficient mice) produced significant enhancement of inotropic and lusitropic effects on left ventricular function and improved cardiac output. These results demonstrate the potent cardiovascular physiologic actions of UcnII in both wild-type and cardiomyopathic mice and support a potential beneficial use of this peptide in therapy of congestive heart failure.
Assuntos
Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Hormônio Liberador da Corticotropina/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Hormônio Liberador da Corticotropina/administração & dosagem , Insuficiência Cardíaca/genética , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Contração Miocárdica/efeitos dos fármacos , Receptores de Hormônio Liberador da Corticotropina/deficiência , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Urocortinas , Resistência Vascular/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
The tyrosine kinase receptor erbB2, also known in humans as Her2, is a member of the epidermal growth factor receptor (EGFR or erbB1) family, which also includes erbB3 and erbB4. The erbBs were discovered in an avian erythroblastosis tumor virus and exhibited similarities to human EGFR (Yarden and Sliwkowski, 2001). Her2/erbB2 is highly expressed in many cancer types. Its overexpression is correlated with a poor prognosis for breast and ovarian cancer patients. ErbB receptors bind to a family of growth factors, termed neuregulins/heregulin (NRG/HRG), which comprise NRG-1, -2, -3, and -4 and include multiple isoforms. ErbB2/Her2 is an orphan receptor that does not bind ligand alone but heterodimerizes with the other erbB receptors for NRG signaling. ErbB2 is expressed in multiple neuronal and non-neuronal tissues in embryos and adult animals, including the heart. Genetic data demonstrated that erbB2 is required for normal embryonic development of neural crest-derived cranial sensory neurons. ErbB2/Her2-null mutant embryos of a trabeculation defect die before embryonic day (E) 11. To study its role at later stages of development, we generated a transgenic mouse line that specifically expresses the rat erbB2 cDNA in the heart under the control of the cardiac-specific alpha-myosin heavy chain promoter. When crossed into the null background, the expression of the rat erbB2 cDNA rescued the cardiac phenotype in the erbB2-null mutant mice that survive until birth but display an absence of Schwann cells and a severe loss of both motor and spinal sensory neurons. To study the role of erbB2 in the adult heart, we generated conditional mutant mice carrying a cardiac-restricted deletion of erbB2. These erbB2 conditional mutants exhibited multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning, and decreased contractility. Interestingly, treatment of breast cancers overexpressing erbB2 with Herceptin (Trastuzumab), a humanized monoclonal antibody specific to the extracellular domain of erbB2, results in some patients developing cardiac dysfunction. The adverse effect is increased significantly in those patients who also receive the chemotherapeutical agent anthracycline. We found that erbB2-deficient cardiac myocytes are more susceptible to anthracycline-induced cytotoxicity. These results suggest that erbB2 signaling in the heart is essential for the prevention of dilated cardiomyopathy. These lines of mice provide models with which to elucidate the molecular and cellular mechanisms by which erbB2 signaling regulates cardiac functions. These mice also will provide important information for devising strategies to mitigate the cardiotoxic effects of Herceptin treatment, allowing for the potential expanded use of this drug to treat all cancers overexpressing erbB2.
Assuntos
Coração/crescimento & desenvolvimento , Coração/fisiologia , Receptor ErbB-2/fisiologia , Animais , Cardiomegalia , Sobrevivência Celular , Humanos , Neovascularização Fisiológica , Neurregulinas/fisiologia , Transdução de SinaisRESUMO
We utilized the Cre-LoxP system to establish erbB2 conditional mutant mice in order to investigate the role of erbB2 in postnatal development of the enteric nervous system. The erbB2/nestin-Cre conditional mutants exhibit retarded growth, distended colons, and premature death, resembling human Hirschsprung's disease. Enteric neurons and glia are present at birth in the colon of erbB2/nestin-Cre mutants; however, a marked loss of multiple classes of enteric neurons and glia occurs by 3 weeks of age. Furthermore, we demonstrate that the requirement for erbB2 in maintaining the enteric nervous system is not cell autonomous, but rather erbB2 signaling in the colonic epithelia is required for the postnatal survival of enteric neurons and glia.
Assuntos
Comunicação Celular/genética , Sobrevivência Celular/genética , Colo/crescimento & desenvolvimento , Colo/inervação , Sistema Nervoso Entérico/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Proteínas do Tecido Nervoso , Receptor ErbB-2/deficiência , Animais , Animais Recém-Nascidos , Quimera , Colo/citologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/citologia , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/fisiopatologia , Integrases/genética , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Mutantes , Mutagênese Sítio-Dirigida/genética , Mutação/genética , Nestina , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptor ErbB-2/genética , Transgenes/genética , Proteínas Virais/genéticaRESUMO
Developing axons are guided to their targets by chemoattractive and chemorepulsive ligands. Ledda et al., in this issue of Neuron, demonstrate that the target-derived receptor glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1) can also act in trans as an axon guidance molecule for neurons.
Assuntos
Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Quimiotaxia/fisiologia , Proteínas de Drosophila , Cones de Crescimento/metabolismo , Sistema Nervoso/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Sinais (Psicologia) , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Fatores de Crescimento Neural/metabolismo , Sistema Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-retRESUMO
Mice harboring targeted mutations in neuregulin-1 and its receptors (erbB2, erbB3, and erbB4) have been invaluable tools for testing the roles of these genes in vivo as well as for identifying unexpected functions for this signaling system. This review summarizes the advances in understanding the myriad functions of neuregulins in the nervous and neuroendocrine systems that have been revealed by examining gene-targeted mice.