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BACKGROUND: The indwelling nasogastric tube is commonly used for supplying enteral nutrition to patients who are unable to feed themselves, and accurate positioning is essential in the indwelling nasogastric tube in the body of the aforementioned patients. In clinical practice, abdominal radiography, auscultation, and clinical determination of the pH of the gastric juice are routinely used by medical personnel to determine the position of the tube; however, those treatments have proved limitations in specific cases. There are few case reports on the precise positioning of the nasogastric tube in patients with coronavirus disease 2019 (COVID-19), for whom a supply of necessary nutrition support is significant throughout the process of treatment. CASE SUMMARY: A 79-year-old patient, diagnosed with COVID-19 at the stage of combined syndromes with severe bacterial lung infection, respiratory failure, multiple co-morbidities, and a poor nutritional status, was presented to us and required an indwelling nasogastric tube for enteral nutrition support. After pre-treatment assessments including observation of the patient's nasal feeding status and examination of the nasal septal deviation, inflammation, obstruction, nasal leakage of cerebrospinal fluid, and other disorders that might render intubation inappropriate, we measured and marked the length of the nasogastric tube to be placed and delivered the tube to the intended length in the standard manner. Then further scrutiny was conducted to ensure that the tube was not coiled in the mouth, and gentle movements were made to avoid damage to the esophageal mucosa. However, back draw of the gastric juice using an empty needle failed, and the stethoscope could not be used for auscultation due to the specific condition presented by the internal organs of the patient, and the end of the tube was placed in saline with no bubbles spilling out. Therefore, it was not possible to determine whether the nasogastric tube was placed exactly in the stomach and no nutrient infusion was performed for the time being. Subsequently, the ultrasound probe was utilized to view the condition of the patient's stomach, where the nasogastric tube was found to be translucent and running parallel to the esophagus shaped as "=". The pre-conditions were achieved and 100 mL nutritional fluid was fed to the patient, who did not experience any discomfort throughout the procedure. His vital signs were stable with no adverse effects. CONCLUSION: We achieved successfully used ultrasound to position the nasogastric tube in a 79-year-old patient with COVID-19. The repeatable ultrasound application does not involve radiation and causes less disturbance in the neck, making it advantageous for rapid positioning of the nasogastric tube and worthy of clinical promotion and application.
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Purpose: Yap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs). Methods: Lens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism. Results: The heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/- LEC, and overexpression of Crim1 restored Yap1+/- LEC cell proliferation in vitro. Conclusions: Homozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Catarata/fisiopatologia , Proteínas de Ciclo Celular/fisiologia , Células Epiteliais/metabolismo , Animais , Western Blotting , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Bromodesoxiuridina/metabolismo , Catarata/metabolismo , Contagem de Células , Proliferação de Células/fisiologia , Células Cultivadas , Progressão da Doença , Células Epiteliais/patologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Heterozigoto , Marcação In Situ das Extremidades Cortadas , Cristalino/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Sinalização YAPRESUMO
Nuclear YAP1 plays a critical role in regulation of stem cell proliferation, tissue regeneration, and organ size in many types of epithelia. Due to rapid turnover of most epithelial cell types, the cytoplasmic function of YAP1 in epithelial cells has not been well studied. The retinal pigment epithelium (RPE) is a highly polarized epithelial cell type maintained at a senescence state, and offers an ideal cell model to study the active role of YAP1 in maintenance of the adult epithelial phenotype. Here, we show that the cytoplasmic function of YAP1 is essential to maintain adult RPE differentiation. Knockout of Yap1 in the adult mouse RPE caused cell depolarization and tight junction breakdown, and led to inhibition of RPE65 expression, diminishment of RPE pigments, and retraction of microvilli and basal infoldings. These changes in RPE further prompted the loss of adjacent photoreceptor outer segments and photoreceptor death, which eventually led to decline of visual function in older mice between 6 and 12 months of age. Furthermore, nuclear ß-catenin and its activity were significantly increased in mutant RPE. These results suggest that YAP1 plays an important role in active inhibition of Wnt/ß-catenin signaling, and is essential for downregulation of ß-catenin nuclear activity and prevention of dedifferentiation of adult RPE.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bestrofinas/fisiologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Epitélio Pigmentado da Retina/citologia , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Sinalização YAPRESUMO
Retinal detachment (RD) induces ischemia and oxygen deficiency in the retina and results in multiple pathological events; photoreceptor cell degeneration and death is the eventual cause of vision decline. In this study, we investigated the therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-Exos) in a rat retinal detachment (RD) model. The model was developed using a subretinal injection of 1% hyaluronic acid in male Sprague-Dawley rats. MSC-Exos were sub-retinally injected at the time of retinal separation to study their therapeutic function. The retinal expression levels of inflammatory cytokines TNF-α, IL-1ß, and MCP-1 were detected by RT-PCR, the autophagy-related protein 5 (Atg5) and microtubule-associated protein 1 light chain 3 beta (LC3) were detected by Western blot, and apoptosis was examined using TUNEL assays at 3 days following RD. Retinal structure was observed at 7 days post-RD. Proteomic analysis was also performed to detect proteins carried by MSC-Exos using iTRAQ-based technology combined with one-dimensional nano LC-nano-ESI- MS/MS. We found that expression of TNF-α and IL-1ß were significantly reduced, the LC3-II to LC3-I ratio was enhanced and cleavage of Atg5 was decreased after MSC-Exo treatment. Treatment with MSC-Exos also suppressed photoreceptor cell apoptosis and maintained normal retinal structure when compared to control groups. Proteomic analysis revealed that MSC-Exos contained proteins with anti-inflammatory, neuroprotective and anti-apoptotic effects. These results suggest that MSC-Exos have therapeutic effects on RD-induced retinal injury and can be used to reduce effects of retinal detachment on photoreceptor cell degeneration in patients.
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Exossomos/fisiologia , Células-Tronco Mesenquimais/citologia , Descolamento Retiniano/terapia , Animais , Apoptose/fisiologia , Proteína 5 Relacionada à Autofagia/metabolismo , Western Blotting , Quimiocina CCL2/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Intraoculares , Interleucina-1beta/genética , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras/patologia , Proteômica , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/genéticaRESUMO
Although accumulated evidence supports the notion that mesenchymal stem cells (MSCs) act in a paracrine manner, the mechanisms are still not fully understood. Recently, MSC-derived exosomes (MSC-Exos), a type of microvesicle released from MSCs, were thought to carry functional proteins and RNAs to recipient cells and play therapeutic roles. In the present study, we intravitreally injected MSCs derived from either mouse adipose tissue or human umbilical cord, and their exosomes to observe and compare their functions in a mouse model of laser-induced retinal injury. We found that both MSCs and their exosomes reduced damage, inhibited apoptosis, and suppressed inflammatory responses to obtain better visual function to nearly the same extent in vivo. Obvious down-regulation of monocyte chemotactic protein (MCP)-1 in the retina was found after MSC-Exos injection. In vitro, MSC-Exos also down-regulated MCP-1 mRNA expression in primarily cultured retinal cells after thermal injury. It was further demonstrated that intravitreal injection of an MCP-1-neutralizing antibody promoted the recovery of retinal laser injury, whereas the therapeutic effect of exosomes was abolished when MSC-Exos and MCP-1 were administrated simultaneously. Collectively, these results suggest that MSC-Exos ameliorate laser-induced retinal injury partially through down-regulation of MCP-1.
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Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem da Célula , Epitélio Corneano/citologia , Queratina-12/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco/citologia , Animais , Proteínas de Ciclo Celular , Epitélio Corneano/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Células-Tronco/metabolismo , Cicatrização , Proteínas de Sinalização YAPRESUMO
The inhibition of NF-κB by genetic deletion or pharmacological inhibition of IKK2 significantly reduces laser-induced choroid neovascularization (CNV). To achieve a sustained and controlled intraocular release of a selective and potent IKK2 inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) (MW: 279.29), we developed a biodegradable poly-lactide-co-glycolide (PLGA) polymer-delivery system to further investigate the anti-neovascularization effects of IKK2 inhibition and in vivo biosafety using laser-induced CNV mouse model. The solvent-evaporation method produced spherical TPCA-1-loaded PLGA microparticles characterized with a mean diameter of 2.4 »m and loading efficiency of 80%. Retrobulbar administration of the TPCA-1-loaded PLGA microparticles maintained a sustained drug level in the retina during the study period. No detectable TPCA-1 level was observed in the untreated contralateral eye. The anti-CNV effect of retrobulbarly administrated TPCA-1-loaded PLGA microparticles was assessed by retinal fluorescein leakage and isolectin staining methods, showing significantly reduced CNV development on day 7 after laser injury. Macrophage infiltration into the laser lesion was attenuated as assayed by choroid/RPE flat-mount staining with anti-F4/80 antibody. Consistently, laser induced expressions of Vegfa and Ccl2 were inhibited by the TPCA-1-loaded PLGA treatment. This TPCA-1 delivery system did not cause any noticeable cellular or functional toxicity to the treated eyes as evaluated by histology and optokinetic reflex (OKR) tests; and no systemic toxicity was observed. We conclude that retrobulbar injection of the small-molecule IKK2 inhibitor TPCA-1, delivered by biodegradable PLGA microparticles, can achieve a sustained and controllable drug release into choroid/retina and attenuate laser-induced CNV development without causing apparent systemic toxicity. Our results suggest a potential clinical application of TPCA-1 delivered by microparticles in treatment of CNV in the patients with age-related macular degeneration and other retinal neovascularization diseases.
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Amidas/administração & dosagem , Neovascularização de Coroide/tratamento farmacológico , Quinase I-kappa B/antagonistas & inibidores , Ácido Láctico/administração & dosagem , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Ácido Poliglicólico/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Tiofenos/administração & dosagem , Amidas/química , Animais , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Ácido Láctico/química , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Inibidores de Proteínas Quinases/química , Tiofenos/químicaRESUMO
The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In this study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate-induced tumors from Er/+ skin. Furthermore, short hairpin RNA (shRNA) knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length wild type (WT) or the mutant form found in Er/Er mice. However, Er 14-3-3σ does not interact with Yap1, as demonstrated by coimmunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, and thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in the Er/Er epidermis.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Queratinócitos/metabolismo , Fosfoproteínas/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Ciclo Celular , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Epiderme/metabolismo , Regulação da Expressão Gênica , Heterozigoto , Homozigoto , Queratinócitos/citologia , Lentivirus/genética , Camundongos , Fenótipo , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Pele/metabolismo , Acetato de Tetradecanoilforbol , Proteínas de Sinalização YAPRESUMO
Pathological retinal neovascularization, including retinopathy of prematurity and age-related macular degeneration, is the most common cause of blindness worldwide. Insulin-like growth factor-1 (IGF-1) has a direct mitogenic effect on endothelial cells, which is the basis of angiogenesis. Vascular endothelial growth factor (VEGF) activation in response to IGF-1 is well documented; however, the molecular mechanisms responsible for the termination of IGF-1 signaling are still not completely elucidated. Here, we show that the polypyrimidine tract-binding protein-associated splicing factor (PSF) is a potential negative regulator of VEGF expression induced by IGF stimulation. Functional analysis demonstrated that ectopic expression of PSF inhibits IGF-1-stimulated transcriptional activation and mRNA expression of the VEGF gene, whereas knockdown of PSF increased IGF-1-stimulated responses. PSF recruited Hakai to the VEGF transcription complex, resulting in inhibition of IGF-1-mediated transcription. Transfection with Hakai siRNA reversed the PSF-mediated transcriptional repression of VEGF gene transcription. In summary, these results show that PSF can repress the transcriptional activation of VEGF stimulated by IGF-1 via recruitment of the Hakai complex and delineate a novel regulatory mechanism of IGF-1/VEGF signaling that may have implications in the pathogenesis of neovascularization in ocular diseases.
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Fator de Crescimento Insulin-Like I/metabolismo , Oxigênio/efeitos adversos , Proteínas de Ligação a RNA/metabolismo , Neovascularização Retiniana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Fator de Crescimento Insulin-Like I/genética , Masculino , Camundongos , Oxigênio/farmacologia , Fator de Processamento Associado a PTB , Proteínas de Ligação a RNA/genética , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Phagocytic clearance of the spent photoreceptor outer segments (OS) by RPE cells is regulated by circadian rhythm cycle and is essential for photoreceptor integrity and function. Mertk regulates RPE phagocytosis and a deficiency in Mertk causes photoreceptor degeneration and visual loss. This study aimed to investigate Mertk regulation of the microRNAs (miRNA), potentially regulating expression of their target genes, which affect phagocytosis. The differentially expressed miRNAs were identified using miRCURY(TM) microRNA Arrays from total RNA isolated at 0900 h and 1900 h from the mechanically dissociated RPE sheets of the WT and Mertk (-/-) mice, which were housed in a 12-h light-dark cycle with the lighting onset at 0700 h (7:00am). Validation of the differentially expressed miRNAs and assessment of the putative miRNA target gene expression were performed by real-time PCR. Among the differentially expressed miRNAs in the Mertk (-/-) RPE, seven miRNAs were up-regulated and 13 were down-regulated in the morning groups. Similarly, 24 miRNAs were found to be up-regulated and 13 were down-regulated in the evening groups. To search for those that may participate in regulating expression of cytoskeletal proteins, we examined the predicted target genes that might participate in phagocytosis were examined by real-time PCR. Of nine potential altered targets, four deregulated genes were myosin subunits. Notably, multiple members of the 21 up-regulated miRNAs can theoretically recognize these down-regulated mRNAs, particularly MyH14 and Myl3. This study shows that loss of Mertk alters miRNA expression, which in turn affects expression of the downstream target genes, potentially affecting phagocytosis.
Assuntos
MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas/deficiência , Receptores Proteína Tirosina Quinases/deficiência , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina QuinaseRESUMO
The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. Here, we show that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppresses Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promotes tissue growth when apoptosis is prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. Thus, our results have identified a novel molecular link between the Hippo and JNK pathways and implicated the essential role of the JNK pathway in Hippo signaling-related tumorigenesis.
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Proteínas de Drosophila/metabolismo , Discos Imaginais/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Asas de Animais/embriologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Discos Imaginais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Asas de Animais/citologia , Proteínas de Sinalização YAP , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The Tyro3, Axl and Mertk (TAM) subfamily of receptor protein tyrosine kinases functions in cell growth, differentiation, survival, and most recently found, in the regulation of immune responses and phagocytosis. All three receptors and their ligands, Gas6 (growth arrest-specific gene 6) and protein S, are expressed in the central nervous system (CNS). TAM receptors play pivotal roles in adult hippocampal neurogenesis. Loss of these receptors causes a comprised neurogenesis in the dentate gyrus of adult hippocampus. TAM receptors have a negative regulatory effect on microglia and peripheral antigen-presenting cells, and play a critical role in preventing overproduction of pro-inflammatory cytokines detrimental to the proliferation, differentiation, and survival of adult neuronal stem cells (NSCs). Besides, these receptors also play an intrinsic trophic function in supporting NSC survival, proliferation, and differentiation into immature neurons. All these events collectively ensure a sustained neurogenesis in adult hippocampus.
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Hipocampo/citologia , Hipocampo/metabolismo , Neurogênese/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Fatores Etários , Animais , Humanos , Transdução de Sinais/fisiologiaRESUMO
Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (ß-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.
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Células-Tronco Neurais/metabolismo , Neurogênese/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Apoptose/genética , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptor trkA/biossíntese , Receptor trkB/biossíntese , Receptor trkC/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Proteínas Recombinantes , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2(-/flox) mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.
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Neovascularização de Coroide/terapia , Quinase I-kappa B/antagonistas & inibidores , Animais , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Lasers , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Transdução de SinaisRESUMO
TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1ß, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.
Assuntos
Giro Denteado/patologia , Microglia/fisiologia , Neurogênese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Citocinas/genética , Replicação do DNA , Encefalite/imunologia , Encefalite/patologia , Regulação da Expressão Gênica , Indometacina/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/deficiência , Interleucina-6/genética , Interleucina-6/fisiologia , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , NF-kappa B/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptores Toll-Like/imunologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
The Tyro3, Axl and Mertk (TAM) triply knockout (TKO) mice exhibit systemic autoimmune diseases, with characteristics of increased proinflammatory cytokine production, autoantibody deposition and autoreactive lymphocyte infiltration into a variety of tissues. Here we show that TKO mice produce high level of serum TNF-α and specific autoantibodies deposited onto brain blood vessels. The brain-blood barrier (BBB) in mutant brains exhibited increased permeability for Evans blue and fluorescent-dextran, suggesting a breakdown of the BBB in the mutant brains. Impaired BBB integrity facilitated autoreactive T cells infiltrating into all regions of the mutant brains. Brain autoimmune disorder caused accumulation of the ubiquitin-reactive aggregates in the mutant hippocampus, and early formation of autofluorescent lipofuscins in the neurons throughout the entire brains. Chronic neuroinflammation caused damage of the hippocampal mossy fibers and neuronal apoptotic death. This study shows that chronic systemic inflammation and autoimmune disorders in the TKO mice cause neuronal damage and death.
Assuntos
Apoptose , Doenças Autoimunes/genética , Dano Encefálico Crônico/imunologia , Região CA3 Hipocampal/patologia , Neurônios/fisiologia , Animais , Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Barreira Hematoencefálica/metabolismo , Dano Encefálico Crônico/genética , Dano Encefálico Crônico/patologia , Região CA3 Hipocampal/irrigação sanguínea , Região CA3 Hipocampal/imunologia , Permeabilidade Capilar/imunologia , Células Cultivadas , Citocinas/metabolismo , Giro Denteado/irrigação sanguínea , Giro Denteado/imunologia , Giro Denteado/patologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Corpos de Inclusão/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/imunologia , Microvasos/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue , Proteínas Ubiquitinadas/metabolismo , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
PURPOSE: The Notch signaling pathway plays crucial roles in regulation of cell proliferation, differentiation and cell fate decision in multiple tissues and cell types. This study was designed to test the effects of enhanced Notch activity on corneal epithelium homeostasis and wound healing using the transgenic mice that overexpressed an activated Notch1 (NICD) in cornea epithelium. METHODS: The studies were performed on R26(fN1-ICD) transgenic mice that carry a NICD cDNA (cDNA) whose expression is prevented by a "Lox-STOP-Lox" cassette. When this transgenic mouse is bred to a mouse strain carrying a Cre recombinase expression cassette driven by a tissue-specific keratin 14 (K14) promoter, the floxed "STOP" cassette is excised and NICD is expressed in the cornea epithelium. The expression level of NICD and its downstream target genes, hairy and enhancer of split 1 (Hes1) and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), in the transgenic corneal epithelium was examined by quantitative PCR (qPCR). The phenotypes and morphology of the transgenic corneal epithelium were compared with that of wild type (WT) controls. The proliferation rate of the epithelial cells was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the differentiation statues were examined by K14, tumor protein p63 (p63), K12, and zona occludens 1 (ZO-1) immunoreactivity at either normal developmental condition or after corneal epithelial debridement. The corneal epithelial response to wound healing was studied by fluorescent staining and Richardson's staining macroscopically and by H&E staining at microscope level at 0, 6, 12, 18, and 24 h post injury. RESULTS: Although overexpression of NICD in cornea epithelium led to upregulation of its downstream targets, i.e., Hes1 and Hey1, this did not alter corneal epithelial cell proliferation and differentiation. However, wound healing induced Notch activity and overexpression of NICD promoted corneal epithelial wound healing, which was in agreement with more rapid early proliferation response in NICD transgenic mice than in the wild type control mice. CONCLUSIONS: These findings further demonstrate the functional role of Notch signaling in corneal epithelium wound healing response.
Assuntos
Epitélio Corneano/lesões , Epitélio Corneano/fisiopatologia , Receptor Notch1/fisiologia , Cicatrização/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Primers do DNA/genética , Epitélio Corneano/patologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Homeostase , Camundongos , Camundongos Transgênicos , Receptor Notch1/genética , Transdução de Sinais , Fatores de Transcrição HES-1 , Cicatrização/genéticaRESUMO
Although the physiological basis of canonical or classical IκB kinase ß (IKKß)-nuclear factor κB (NF-κB) signaling pathway is well established, how alternative NF-κB signaling functions beyond its role in lymphoid development remains unclear. In particular, alternative NF-κB signaling has been linked with cellular metabolism, but this relationship is poorly understood. In this study, we show that mice deleted for the alternative NF-κB components IKKα or RelB have reduced mitochondrial content and function. Conversely, expressing alternative, but not classical, NF-κB pathway components in skeletal muscle stimulates mitochondrial biogenesis and specifies slow twitch fibers, suggesting that oxidative metabolism in muscle is selectively controlled by the alternative pathway. The alternative NF-κB pathway mediates this specificity by direct transcriptional activation of the mitochondrial regulator PPAR-γ coactivator 1ß (PGC-1ß) but not PGC-1α. Regulation of PGC-1ß by IKKα/RelB also is mammalian target of rapamycin (mTOR) dependent, highlighting a cross talk between mTOR and NF-κB in muscle metabolism. Together, these data provide insight on PGC-1ß regulation during skeletal myogenesis and reveal a unique function of alternative NF-κB signaling in promoting an oxidative metabolic phenotype.
Assuntos
Respiração Celular , Quinase I-kappa B/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , NF-kappa B/metabolismo , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Mioblastos/citologia , NF-kappa B/genética , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using 'repeated epilation' heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.
Assuntos
Proteínas 14-3-3/fisiologia , Transformação Celular Neoplásica , Fosfoproteínas/fisiologia , Neoplasias Cutâneas/fisiopatologia , Transativadores/fisiologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Western Blotting , Carcinógenos/toxicidade , Diferenciação Celular , Proliferação de Células , Inativação Gênica , Homeostase , Queratinócitos/citologia , Camundongos , Camundongos Mutantes , Fosfoproteínas/genética , Reação em Cadeia da Polimerase , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidade , Transativadores/genéticaRESUMO
The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.