RESUMO
The presence of basal lineage characteristics signifies hyperaggressive human adenocarcinomas of the breast, bladder and pancreas. However, the biochemical mechanisms that maintain this aberrant cell state are poorly understood. Here we performed marker-based genetic screens in search of factors needed to maintain basal identity in pancreatic ductal adenocarcinoma (PDAC). This approach revealed MED12 as a powerful regulator of the basal cell state in this disease. Using biochemical reconstitution and epigenomics, we show that MED12 carries out this function by bridging the transcription factor ΔNp63, a known master regulator of the basal lineage, with the Mediator complex to activate lineage-specific enhancer elements. Consistent with this finding, the growth of basal-like PDAC is hypersensitive to MED12 loss when compared to PDAC cells lacking basal characteristics. Taken together, our genetic screens have revealed a biochemical interaction that sustains basal identity in human cancer, which could serve as a target for tumor lineage-directed therapeutics.
Assuntos
Carcinoma Ductal Pancreático , Complexo Mediador , Neoplasias Pancreáticas , Fatores de Transcrição , Proteínas Supressoras de Tumor , Humanos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Linhagem da Célula/genética , Elementos Facilitadores GenéticosRESUMO
Exposure to pathogens throughout a lifetime influences immunity and organ function. Here, we explore how the systemic host-response to bacterial urinary tract infection (UTI) induces tissue-specific alterations to the mammary gland. Utilizing a combination of histological tissue analysis, single cell transcriptomics, and flow cytometry, we identify that mammary tissue from UTI-bearing mice displays collagen deposition, enlarged ductal structures, ductal hyperplasia with atypical epithelial transcriptomes and altered immune composition. Bacterial cells are absent in the mammary tissue and blood of UTI-bearing mice, therefore, alterations to the distal mammary tissue are mediated by the systemic host response to local infection. Furthermore, broad spectrum antibiotic treatment resolves the infection and restores mammary cellular and tissue homeostasis. Systemically, unresolved UTI correlates with increased plasma levels of the metalloproteinase inhibitor, TIMP1, which controls extracellular matrix remodeling and neutrophil function. Treatment of nulliparous and post-lactation UTI-bearing female mice with a TIMP1 neutralizing antibody, restores mammary tissue normal homeostasis, thus providing evidence for a link between the systemic host response during UTI and mammary gland alterations.
Assuntos
Glândulas Mamárias Animais , Infecções Urinárias , Animais , Feminino , Camundongos , Colágeno , Matriz Extracelular/fisiologia , HomeostaseRESUMO
Chronic stress is associated with increased risk of metastasis and poor survival in cancer patients, yet the reasons are unclear. We show that chronic stress increases lung metastasis from disseminated cancer cells 2- to 4-fold in mice. Chronic stress significantly alters the lung microenvironment, with fibronectin accumulation, reduced T cell infiltration, and increased neutrophil infiltration. Depleting neutrophils abolishes stress-induced metastasis. Chronic stress shifts normal circadian rhythm of neutrophils and causes increased neutrophil extracellular trap (NET) formation via glucocorticoid release. In mice with neutrophil-specific glucocorticoid receptor deletion, chronic stress fails to increase NETs and metastasis. Furthermore, digesting NETs with DNase I prevents chronic stress-induced metastasis. Together, our data show that glucocorticoids released during chronic stress cause NET formation and establish a metastasis-promoting microenvironment. Therefore, NETs could be targets for preventing metastatic recurrence in cancer patients, many of whom will experience chronic stress due to their disease.
Assuntos
Armadilhas Extracelulares , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neutrófilos/patologia , Neoplasias Pulmonares/patologia , Pulmão/patologia , Microambiente TumoralRESUMO
Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.
Assuntos
Melanoma , Linfócitos T , Camundongos , Animais , Linfócitos T/patologia , Neutrófilos/patologia , Deriva e Deslocamento Antigênicos , Imunoterapia , Antígeno CTLA-4RESUMO
Neutrophils are major effectors and regulators of the immune system. They play critical roles not only in the eradication of pathogens but also in cancer initiation and progression. Conversely, the presence of cancer affects neutrophil activity, maturation, and lifespan. By promoting or repressing key neutrophil functions, cancer cells co-opt neutrophil biology to their advantage. This co-opting includes hijacking one of neutrophils' most striking pathogen defense mechanisms: the formation of neutrophil extracellular traps (NETs). NETs are web-like filamentous extracellular structures of DNA, histones, and cytotoxic granule-derived proteins. Here, we discuss the bidirectional interplay by which cancer stimulates NET formation, and NETs in turn support disease progression. We review how vascular dysfunction and thrombosis caused by neutrophils and NETs underlie an elevated risk of death from cardiovascular events in cancer patients. Finally, we propose therapeutic strategies that may be effective in targeting NETs in the clinical setting.
Assuntos
Armadilhas Extracelulares , Neoplasias , Trombose , Humanos , Armadilhas Extracelulares/metabolismo , Neutrófilos , Histonas/metabolismo , Trombose/etiologia , Trombose/metabolismo , DNA/metabolismo , Neoplasias/metabolismoRESUMO
The EWS-FLI1 fusion oncoprotein deregulates transcription to initiate the paediatric cancer Ewing sarcoma. Here we used a domain-focused CRISPR screen to implicate the transcriptional repressor ETV6 as a unique dependency in this tumour. Using biochemical assays and epigenomics, we show that ETV6 competes with EWS-FLI1 for binding to select DNA elements enriched for short GGAA repeat sequences. Upon inactivating ETV6, EWS-FLI1 overtakes and hyper-activates these cis-elements to promote mesenchymal differentiation, with SOX11 being a key downstream target. We show that squelching of ETV6 with a dominant-interfering peptide phenocopies these effects and suppresses Ewing sarcoma growth in vivo. These findings reveal targeting of ETV6 as a strategy for neutralizing the EWS-FLI1 oncoprotein by reprogramming of genomic occupancy.
Assuntos
Sarcoma de Ewing , Criança , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Acute lung injury (ALI) can cause acute respiratory distress syndrome (ARDS), a lethal condition with limited treatment options and currently a common global cause of death due to COVID-19. ARDS secondary to transfusion-related ALI (TRALI) has been recapitulated preclinically by anti-MHC-I antibody administration to LPS-primed mice. In this model, we demonstrate that inhibitors of PTP1B, a protein tyrosine phosphatase that regulates signaling pathways of fundamental importance to homeostasis and inflammation, prevented lung injury and increased survival. Treatment with PTP1B inhibitors attenuated the aberrant neutrophil function that drives ALI and was associated with release of myeloperoxidase, suppression of neutrophil extracellular trap (NET) formation, and inhibition of neutrophil migration. Mechanistically, reduced signaling through the CXCR4 chemokine receptor, particularly to the activation of PI3Kγ/AKT/mTOR, was essential for these effects, linking PTP1B inhibition to promoting an aged-neutrophil phenotype. Considering that dysregulated activation of neutrophils has been implicated in sepsis and causes collateral tissue damage, we demonstrate that PTP1B inhibitors improved survival and ameliorated lung injury in an LPS-induced sepsis model and improved survival in the cecal ligation and puncture-induced (CLP-induced) sepsis model. Our data highlight the potential for PTP1B inhibition to prevent ALI and ARDS from multiple etiologies.
Assuntos
Lesão Pulmonar Aguda , COVID-19 , Síndrome do Desconforto Respiratório , Sepse , Lesão Pulmonar Aguda/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Neutrófilos , Síndrome do Desconforto Respiratório/etiologia , Sepse/complicaçõesRESUMO
Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.
Assuntos
Linhagem da Célula , Neoplasias Pulmonares , Proteínas de Neoplasias , Fatores de Transcrição de Octâmero , Carcinoma de Pequenas Células do Pulmão , Animais , Humanos , Camundongos , Neoplasias Pulmonares/patologia , Mucosa/patologia , Família Multigênica/genética , Proteínas de Neoplasias/metabolismo , Motivos de Nucleotídeos , Fatores de Transcrição de Octâmero/metabolismo , Fatores do Domínio POU/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , TransativadoresRESUMO
The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts inâ situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells inâ vitro and for direct imaging of chemotherapy-induced apoptosis inâ vivo in mouse models of breast cancer.
Assuntos
Corantes FluorescentesRESUMO
The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts inâ situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells inâ vitro and for direct imaging of chemotherapy-induced apoptosis inâ vivo in mouse models of breast cancer.
RESUMO
Tumor-associated macrophages (TAMs) promote metastasis and inhibit T cells, but macrophages can be polarized to kill cancer cells. Macrophage polarization could thus be a strategy for controlling cancer. We show that macrophages from metastatic pleural effusions of breast cancer patients can be polarized to kill cancer cells with monophosphoryl lipid A (MPLA) and interferon (IFN) γ. MPLA + IFNγ injected intratumorally or intraperitoneally reduces primary tumor growth and metastasis in breast cancer mouse models, suppresses metastasis, and enhances chemotherapy response in an ovarian cancer model. Both macrophages and T cells are critical for the treatment's anti-metastatic effects. MPLA + IFNγ stimulates type I IFN signaling, reprograms CD206+ TAMs to inducible NO synthase (iNOS)+ macrophages, and activates cytotoxic T cells through macrophage-secreted interleukin-12 (IL-12) and tumor necrosis factor alpha (TNFα). MPLA and IFNγ are used individually in clinical practice and together represent a previously unexplored approach for engaging a systemic anti-tumor immune response.
Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Metástase Neoplásica/imunologia , Animais , Humanos , CamundongosRESUMO
Stress is linked to negative outcomes in cardiovascular diseases but exactly why is unclear. In this issue of Immunity, Xu et al. report that stress elicits glucocorticoid-induced gut permeability, in turn triggering the expansion of a population of neutrophils that can stimulate vaso-occlusive episodes.
Assuntos
Microbioma Gastrointestinal , Microbiota , Doenças Vasculares , Emoções , Humanos , InflamaçãoRESUMO
C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2-/- cancer cells did not induce immune suppression in Batf3-/- mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response.
Assuntos
Imunidade Adaptativa/imunologia , Tolerância Imunológica , Neoplasias Mamárias Experimentais/imunologia , Receptores CCR2/fisiologia , Imunidade Adaptativa/fisiologia , Animais , Apoptose , Antígeno B7-H1/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Tolerância Imunológica/imunologia , Tolerância Imunológica/fisiologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR2/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologiaRESUMO
COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.
Assuntos
Infecções por Coronavirus/complicações , Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Pneumonia Viral/complicações , Trombose/complicações , Adulto , Idoso , Betacoronavirus/imunologia , Plaquetas/imunologia , Plaquetas/patologia , Proteínas Sanguíneas/imunologia , COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Neutrófilos/patologia , Pandemias , Peroxidase/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Estudos Prospectivos , SARS-CoV-2 , Trombose/imunologia , Trombose/patologiaRESUMO
Hypotonic stress causes the activation of swelling-activated nonselective cation channels (NSCCs), which leads to Ca2+-dependent regulatory volume decrease (RVD) and adaptive maintenance of the cell volume; however, the molecular identities of the osmosensitive NSCCs remain unclear. Here, we identified TMEM63B as an osmosensitive NSCC activated by hypotonic stress. TMEM63B is enriched in the inner ear sensory hair cells. Genetic deletion of TMEM63B results in necroptosis of outer hair cells (OHCs) and progressive hearing loss. Mechanistically, the TMEM63B channel mediates hypo-osmolarity-induced Ca2+ influx, which activates Ca2+-dependent K+ channels required for the maintenance of OHC morphology. These findings demonstrate that TMEM63B is an osmosensor of the mammalian inner ear and the long-sought cation channel mediating Ca2+-dependent RVD.
Assuntos
Audição/efeitos dos fármacos , Soluções Hipotônicas/farmacologia , Transporte de Íons/fisiologia , Concentração Osmolar , Canais de Potássio/metabolismo , Animais , Cálcio/metabolismo , Cátions/metabolismo , Tamanho Celular/efeitos dos fármacos , Camundongos Knockout , Potássio/metabolismo , Canais de Potássio/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
A highly aggressive subset of pancreatic ductal adenocarcinomas undergo trans-differentiation into the squamous lineage during disease progression. Here, we investigated whether squamous trans-differentiation of human and mouse pancreatic cancer cells can influence the phenotype of non-neoplastic cells in the tumor microenvironment. Conditioned media experiments revealed that squamous pancreatic cancer cells secrete factors that recruit neutrophils and convert pancreatic stellate cells into cancer-associated fibroblasts (CAFs) that express inflammatory cytokines at high levels. We use gain- and loss-of-function approaches to show that squamous-subtype pancreatic tumor models become enriched with neutrophils and inflammatory CAFs in a p63-dependent manner. These effects occur, at least in part, through p63-mediated activation of enhancers at pro-inflammatory cytokine loci, which includes IL1A and CXCL1 as key targets. Taken together, our findings reveal enhanced tissue inflammation as a consequence of squamous trans-differentiation in pancreatic cancer, thus highlighting an instructive role of tumor cell lineage in reprogramming the stromal microenvironment.
Assuntos
Carcinoma Ductal Pancreático/patologia , Transdiferenciação Celular/fisiologia , Inflamação/patologia , Neoplasias Pancreáticas/patologia , Animais , Fibroblastos Associados a Câncer/fisiologia , Carcinoma Ductal Pancreático/imunologia , Linhagem da Célula , Citocinas/genética , Citocinas/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Neoplasias Pancreáticas/imunologia , Células Estromais/patologia , Microambiente TumoralRESUMO
Stress has long been suspected to negatively influence cancer mortality, yet the molecular mechanisms responsible for this effect have only recently been identified. A new study identifies a stress-induced response in dendritic cells-the activation of the glucocorticoid-inducible transcriptional regulator TSC22D3-as a potent, immunosuppressive effect of stress on cancer.
Assuntos
Células Dendríticas/imunologia , Glucocorticoides/metabolismo , Imunoterapia , Neoplasias/terapia , Estresse Psicológico/imunologia , Células Dendríticas/metabolismo , Progressão da Doença , Glucocorticoides/imunologia , Humanos , Tolerância Imunológica , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias/psicologia , Estresse Psicológico/psicologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Resultado do TratamentoRESUMO
Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a trans-differentiation event in this disease.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/fisiopatologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Humanos , Pulmão/patologia , Camundongos , Receptor IGF Tipo 1/metabolismoRESUMO
The p53 signaling network is indispensible in cellular stress responses and tumor suppression. Negative regulations of p53 by mouse double minute 2 (MDM2) and its homolog MDM4 are an integrated component of the network and have been implicated in regulating the stress responses and the maintenance of normal development and homeostasis of multiple somatic cell lineages. However, the regulatory role of MDM2 on p53 and stress responses in female germ cells remains undetermined. Here, we used the Cre-loxP system to delete Mdm2 in oocytes at different stages of folliculogenesis in mice. Mdm2 deletion resulted in a clear p53 nuclear accumulation in the oocytes and impeded fertilities with early follicular loss in mice, resembling human premature ovarian failure phenotypes. These phenotypes were fully rescued by concurrent deletion of p53 in mice. In addition, Nutlin-3, a small molecule compound that inhibited the binding of MDM2 to p53, also promoted p53-dependent oocyte death. Although cancer therapeutic agents 5-fluorouracil and doxorubicin could not induce a robust p53 activation in the wild-type oocytes, they induced p53 nuclear accumulation in the Mdm2 and Mdm4 double heterozygous oocytes. These results demonstrated a critical prosurvival role for MDM2 in the oocytes. Moreover, they suggested a more tightened and rigorous regulatory mode for the MDM2/MDM4-p53 network in female germ cells under stress situations.
Assuntos
Oócitos/metabolismo , Oogênese/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Western Blotting , Feminino , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/crescimento & desenvolvimentoRESUMO
AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.