RESUMO
The efficacy of immunotherapy is limited by the paucity of T cells delivered and infiltrated into the tumors through aberrant tumor vasculature. Here, we report that phosphoglycerate dehydrogenase (PHGDH)-mediated endothelial cell (EC) metabolism fuels the formation of a hypoxic and immune-hostile vascular microenvironment, driving glioblastoma (GBM) resistance to chimeric antigen receptor (CAR)-T cell immunotherapy. Our metabolome and transcriptome analyses of human and mouse GBM tumors identify that PHGDH expression and serine metabolism are preferentially altered in tumor ECs. Tumor microenvironmental cues induce ATF4-mediated PHGDH expression in ECs, triggering a redox-dependent mechanism that regulates endothelial glycolysis and leads to EC overgrowth. Genetic PHGDH ablation in ECs prunes over-sprouting vasculature, abrogates intratumoral hypoxia, and improves T cell infiltration into the tumors. PHGDH inhibition activates anti-tumor T cell immunity and sensitizes GBM to CAR T therapy. Thus, reprogramming endothelial metabolism by targeting PHGDH may offer a unique opportunity to improve T cell-based immunotherapy.
Assuntos
Glioblastoma , Receptores de Antígenos Quiméricos , Animais , Camundongos , Humanos , Glioblastoma/terapia , Glioblastoma/metabolismo , Fosfoglicerato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva , Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis.
Assuntos
Fibroblastos Associados a Câncer , Melanoma , Neoplasias Pancreáticas , Animais , Fibroblastos Associados a Câncer/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Camundongos , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
Inflammatory breast cancer (IBC) is a highly metastatic breast carcinoma with high frequency of estrogen receptor α (ERα) negativity. Here we explored the role of the second ER subtype, ERß, and report expression in IBC tumors and its correlation with reduced metastasis. Ablation of ERß in IBC cells promoted cell migration and activated gene networks that control actin reorganization, including G-protein-coupled receptors and downstream effectors that activate Rho GTPases. Analysis of preclinical mouse models of IBC revealed decreased metastasis of IBC tumors when ERß was expressed or activated by chemical agonists. Our findings support a tumor-suppressive role of ERß by demonstrating the ability of the receptor to inhibit dissemination of IBC cells and prevent metastasis. On the basis of these findings, we propose ERß as a potentially novel biomarker and therapeutic target that can inhibit IBC metastasis and reduce its associated mortality. SIGNIFICANCE: These findings demonstrate the capacity of ERß to elicit antimetastatic effects in highly aggressive inflammatory breast cancer and propose ERß and the identified associated genes as potential therapeutic targets in this disease.
Assuntos
Actinas/metabolismo , Movimento Celular/genética , Receptor beta de Estrogênio/metabolismo , Neoplasias Inflamatórias Mamárias/metabolismo , Transdução de Sinais/genética , Citoesqueleto de Actina/metabolismo , Animais , Estudos de Coortes , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/patologia , Células MCF-7 , Camundongos , Metástase Neoplásica/genética , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant solid tumors are characterized by aberrant vascularity that fuels the formation of an immune-hostile microenvironment and induces resistance to immunotherapy. Vascular abnormalities may be driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide screening of mesenchymal-like transcriptional activation in human glioblastoma (GBM)-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic reprogramming and aberrant vascularization. PAK4 knockout induces adhesion protein re-expression in ECs, reduces vascular abnormalities, improves T cell infiltration and inhibits GBM growth in mice. Moreover, PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to chimeric antigen receptor-T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer a unique opportunity to recondition the vascular microenvironment and strengthen cancer immunotherapy.
Assuntos
Glioblastoma , Receptores de Antígenos Quiméricos , Quinases Ativadas por p21/metabolismo , Animais , Células Endoteliais/metabolismo , Glioblastoma/genética , Imunoterapia , Camundongos , Receptores de Antígenos Quiméricos/metabolismo , Microambiente Tumoral , Quinases Ativadas por p21/genéticaRESUMO
Therapeutic resistance remains a persistent challenge for patients with malignant tumors. Here, we reveal that endothelial cells (ECs) acquire transformation into mesenchymal stem cell (MSC)-like cells in glioblastoma (GBM), driving tumor resistance to cytotoxic treatment. Transcriptome analysis by RNA sequencing (RNA-seq) revealed that ECs undergo mesenchymal transformation and stemness-like activation in GBM microenvironment. Furthermore, we identified a c-Met-mediated axis that induces ß-catenin phosphorylation at Ser675 and Wnt signaling activation, inducing multidrug resistance-associated protein-1(MRP-1) expression and leading to EC stemness-like activation and chemoresistance. Last, genetic ablation of ß-catenin in ECs overcome GBM tumor resistance to temozolomide (TMZ) chemotherapy in vivo. Combination of Wnt inhibition and TMZ chemotherapy eliminated tumor-associated ECs, inhibited GBM growth, and increased mouse survival. These findings identified a cell plasticity-based, microenvironment-dependent mechanism that controls tumor chemoresistance, and suggest that targeting Wnt/ß-catenin-mediated EC transformation and stemness activation may overcome therapeutic resistance in GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Células-Tronco Mesenquimais , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Temozolomida/farmacologia , Microambiente TumoralRESUMO
: Circulating tumor cells (CTC) are known to be present in the blood of patients with glioblastoma (GBM). Here we report that GBM-derived CTC possess a cancer stem cell (CSC)-like phenotype and contribute to local tumorigenesis and recurrence by the process of self-seeding. Genetic probes showed that mouse GBM-derived CTC exhibited Sox2/ETn transcriptional activation and expressed glioma CSC markers, consistent with robust expression of stemness-associated genes including SOX2, OCT4, and NANOG in human GBM patient-derived samples containing CTC. A transgenic mouse model demonstrated that CTC returned to the primary tumor and generated new tumors with enhanced tumorigenic capacity. These CTCs were resistant to radiotherapy and chemotherapy and to circulation stress-induced cell apoptosis. Single-cell RNA-seq analysis revealed that Wnt activation induced stemness and chemoresistance in CTC. Collectively, these findings identify GBM-derived CTC as CSC-like cells and suggest that targeting Wnt may offer therapeutic opportunities for eliminating these treatment-refractory cells in GBM. SIGNIFICANCE: These findings identify CTCs as an alternative source for in situ tumor invasion and recurrence through local micrometastasis, warranting eradication of systemic "out-of-tumor" CTCs as a promising new therapeutic opportunity for GBM.
Assuntos
Glioma/metabolismo , Glioma/patologia , Células Neoplásicas Circulantes/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imunofenotipagem , Masculino , Camundongos , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fenótipo , Estresse Fisiológico , Proteínas Wnt/metabolismoRESUMO
Angiogenesis is a hallmark of cancer. However, most malignant solid tumors exhibit robust resistance to current anti-angiogenic therapies that primarily target VEGF pathways. Here we report that endothelial-mesenchymal transformation induces glioblastoma (GBM) resistance to anti-angiogenic therapy by downregulating VEGFR-2 expression in tumor-associated endothelial cells (ECs). We show that VEGFR-2 expression is markedly reduced in human and mouse GBM ECs. Transcriptome analysis verifies reduced VEGFR-2 expression in ECs under GBM conditions and shows increased mesenchymal gene expression in these cells. Furthermore, we identify a PDGF/NF-κB/Snail axis that induces mesenchymal transformation and reduces VEGFR-2 expression in ECs. Finally, dual inhibition of VEGFR and PDGFR eliminates tumor-associated ECs and improves animal survival in GBM-bearing mice. Notably, EC-specific knockout of PDGFR-ß sensitizes tumors to VEGF-neutralizing treatment. These findings reveal an endothelial plasticity-mediated mechanism that controls anti-angiogenic therapy resistance, and suggest that vascular de-transformation may offer promising opportunities for anti-vascular therapy in cancer.
Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Galinhas , Imunoprecipitação da Cromatina , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família Snail , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Spatiotemporal regulation of tumor immunity remains largely unexplored. Here we identify a vascular niche that controls alternative macrophage activation in glioblastoma (GBM). We show that tumor-promoting macrophages are spatially proximate to GBM-associated endothelial cells (ECs), permissive for angiocrine-induced macrophage polarization. We identify ECs as one of the major sources for interleukin-6 (IL-6) expression in GBM microenvironment. Furthermore, we reveal that colony-stimulating factor-1 and angiocrine IL-6 induce robust arginase-1 expression and macrophage alternative activation, mediated through peroxisome proliferator-activated receptor-γ-dependent transcriptional activation of hypoxia-inducible factor-2α. Finally, utilizing a genetic murine GBM model, we show that EC-specific knockout of IL-6 inhibits macrophage alternative activation and improves survival in the GBM-bearing mice. These findings illustrate a vascular niche-dependent mechanism for alternative macrophage activation and cancer progression, and suggest that targeting endothelial IL-6 may offer a selective and efficient therapeutic strategy for GBM, and possibly other solid malignant tumors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais/imunologia , Glioblastoma/imunologia , Interleucina-6/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Arginase/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Progressão da Doença , Humanos , Fator Estimulador de Colônias de Macrófagos/imunologia , Camundongos , Microvasos/citologia , Monócitos/imunologia , Neoplasias Experimentais/imunologia , Ativação Transcricional , Microambiente TumoralRESUMO
Cancer stem cells (CSCs) - known to be resistant to genotoxic radiation and chemotherapy - are fundamental to therapy failure and cancer relapse. Here, we reveal that glioma CSCs are hypersensitive to radiation, but a temporal DNA repair mechanism converts the intrinsic sensitivity to genomic instability and treatment resistance. Transcriptome analysis identifies DNA-dependent protein kinase (DNA-PK) as a predominant DNA repair enzyme in CSCs. Notably, DNA-PK activity is suppressed after irradiation when ROS induce the dissociation of DNA-PKcs with Ku70/80, resulting in delayed DNA repair and radiosensitivity; subsequently, after ROS clearance, the accumulated DNA damage and robust activation of DNA-PK induce genomic instability, facilitated by Rad50-mediated cell-cycle arrest, leading to enhanced malignancy, CSC overgrowth, and radioresistance. Finally, we show a requisite in vivo role for DNA-PK in CSC-mediated radioresistance and glioma progression. These findings identify a time-sensitive mechanism controlling CSC resistance to DNA-damaging treatments and suggest DNA-PK/Rad50 as promising targets for CSC eradication.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Instabilidade Genômica/efeitos da radiação , Glioma/radioterapia , Células-Tronco Neoplásicas/efeitos da radiação , Proteínas Nucleares/metabolismo , Tolerância a Radiação/genética , Hidrolases Anidrido Ácido , Animais , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioma/genética , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase-14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor-derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Células Endoteliais/patologia , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Masculino , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , TemozolomidaRESUMO
Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Glicoproteínas/metabolismo , Agregação Plaquetária , Transdução de Sinais/fisiologia , Trombose/metabolismo , Animais , Plaquetas/patologia , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Trombose/genética , Trombose/patologiaRESUMO
Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo. Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.
Assuntos
Apoproteína(a)/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Inflamação/patologia , Metaloproteinase 9 da Matriz/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Animais , Aorta/patologia , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas B/metabolismo , Movimento Celular , Modelos Animais de Doenças , Ativação Enzimática , Fibrinolisina/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Testes de Neutralização , Neutrófilos/enzimologia , Peritonite/patologia , Plasminogênio/deficiência , Plasminogênio/metabolismoRESUMO
Establishing the pattern of expression of transmitters and peptides as well as their receptors in different neuronal types is crucial for understanding the circuitry in various regions of the brain. Previous studies have demonstrated that the transmitter and peptide phenotypes in mouse dorsal spinal cord neurons are determined by the transcription factors Tlx1/3 and Ptf1a. Here we show that these transcription factors also determine the expression of two distinct sets of transmitter and peptide receptor genes in this region. We have screened the expression of 78 receptor genes in the spinal dorsal horn by in situ hybridization. We found that receptor genes Gabra1, Gabra5, Gabrb2, Gria3, Grin3a, Grin3b, Galr1, and Npy1r were preferentially expressed in Tlx3-expressing glutamatergic neurons and their derivatives, and deletion of Tlx1 and Tlx3 resulted in the loss of expression of these receptor genes. Furthermore, we obtained genetic evidence that Tlx3 uses distinct pathways to control the expression of receptor genes. We also found that receptor genes Grm3, Grm4, Grm5, Grik1, Grik2, Grik3, and Sstr2 were mainly expressed in Pax2-expressing GABAergic neurons in the spinal dorsal horn, and their expression in this region was abolished or markedly reduced in Ptf1a and Pax2 deletion mutant mice. Together, our studies indicate that Tlx1/3 and Ptf1a, the key transcription factors for fate determination of glutamatergic and GABAergic neurons in the dorsal spinal cord, are also responsible for controlling the expression of two distinct sets of transmitter and peptide receptor genes.
Assuntos
Proteínas de Homeodomínio/fisiologia , Receptores de Neuropeptídeos/fisiologia , Receptores de Neurotransmissores/fisiologia , Medula Espinal/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Proteínas de Homeodomínio/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Neurotensina/metabolismo , Fator de Transcrição PAX2/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Reação em Cadeia da Polimerase , Receptores da Colecistocinina/genética , Receptores de Glutamato/genética , Receptores de Neuropeptídeos/genética , Receptores de Neurotransmissores/genética , Medula Espinal/metabolismo , Fatores de Transcrição/genética , Proteína Vesicular 1 de Transporte de Glutamato/genética , Ácido gama-Aminobutírico/fisiologiaRESUMO
The involvement of microRNAs (miRNAs) in the development of the neural crest (NC) cells and other neuronal differentiation is still poorly understood. Here, we investigated the global function of miRNAs in embryonic development by examining the Wnt1-cre-mediated Dicer knockout mice. Dicer ablation resulted in malformation of the midbrain and cerebellum and failure of NC and dopaminergic differentiation. First, the Dicer mutant fetuses exhibited dramatic malformation of the tectum and cerebellum and the eyelids were open. Second, the skeletal structures that are derived from the cranial NC were lost or mostly ablated in Dicer mutant mice. Third, deletion of Dicer in the NC cells resulted in the malformation of the dorsal root ganglia, enteric nervous system and sympathetic ganglia. Interestingly, the expression of neuropeptide Y and its potential regulators TrkA, AP-2alpha and AP-2beta was largely abolished in sympathetic neurons of Dicer mutant mice. Fourth, in situ hybridization data revealed that the expression of miR-9, miR-124 and miR-218 in the midbrain and rostral hindbrain area was mostly eliminated in the Dicer mutant mice. We then demonstrated that the development of dopaminergic neurons was impaired in Dicer-deleted mice. Our studies therefore suggest that miRNAs contribute to the embryonic development in multiple locations.