RESUMO
Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.
Assuntos
Deficiência de Ácido Fólico , Defeitos do Tubo Neural , Animais , Camundongos , Ácido Fólico/metabolismo , Actinas/metabolismo , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Polaridade Celular/genética , Fibroblastos/metabolismo , Via de Sinalização Wnt , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Deficiência de Ácido Fólico/metabolismoRESUMO
Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.
Assuntos
Polaridade Celular , Proteínas Desgrenhadas , Proteínas de Drosophila , Proteínas de Membrana , Animais , Camundongos , Polaridade Celular/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Frizzled/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , FosforilaçãoRESUMO
Planar cell polarity (PCP) signaling plays a fundamental role in shaping the development and ongoing function of the nervous system, beginning from early stages of neural tube closure and spanning the maintenance of functional synapses in adults. While mutations in core PCP signaling proteins have long been suspected to underlie neural tube closure defects in humans, recent findings also implicate their potential involvement in neurodevelopmental and neuropsychiatric disorders. Missense and loss-of-function mutations in CELSR3, a core component of PCP signaling complexes, are highly associated with Tourette Disorder. Although the functional significance of these mutations has yet to be elucidated in animal and cell models, the expression patterns of Celsr3 in mice point to alterations in cortico-striato-thalamo-cortical circuits. Here, we briefly review the known functions of Celsr3 for nervous system development. We also propose circuit models for Tourette Disorder by hypothesizing roles for Celsr3 in controlling striatal neuromodulation via effects on cholinergic interneurons, and thalamic inhibition through its functions in thalamic reticular nuclei. Testing these and related hypotheses in animal and cell models will move us closer to unraveling the neuropathogenesis of Tourette Disorder, with the ultimate goal of developing more efficacious treatments for both motor and cognitive symptoms.
Assuntos
Defeitos do Tubo Neural , Síndrome de Tourette , Adulto , Animais , Caderinas/genética , Polaridade Celular/genética , Humanos , Camundongos , Defeitos do Tubo Neural/genética , Neurulação , Receptores de Superfície Celular/genética , Tálamo , Síndrome de Tourette/genéticaRESUMO
OBJECTIVE: To observe the effects of moxibustion on "Shenshu" and "Zusanli" on macrophage polarization and IL-4/STAT6 signaling pathway in rats with rheumatoid arthritis (RA). To further explore the possible anti-inflammatory mechanism of moxibustion in the treatment of RA. METHODS: The rats' right hind paws were injected with freund's complete adjuvant (FCA) to establish the model of RA. Seven days after the injection of FCA, moxibustion therapy was performed on the acupoints of Shenshu (BL23) and Zusanli (ST36) once a day for three weeks. The researchers measured the thickness of the foot pad. ELISA and Histological Analysis were performed to observe the anti-inflammatory effect of moxibustion. Then researchers detected the expression of macrophage phenotype and the expression of IL-4/STAT6 signaling pathway related molecules. RESULTS: It was observed that after the injection of FCA, the rats' feet showed obvious symptoms of redness and swelling. But the symptoms were significantly improved when moxibustion was employed. The study found lower IL-23 and higher IL-4 level in the serum of FCA-injected rats after moxibustion treatment. HE staining showed that the synovium of the RA group was hyperemia and edema, with a large number of inflammatory cells infiltration and vascular dilatation. In the moxibustion group, the degree of synovial hyperemia and edema was improved, and the infiltration of inflammatory cells and vascular dilation were reduced. The study also found that there wer differences among the expressions of macrophage phenotypes in RA, and this was shown by the high expression of CD86 and low expression of CD206. However, the polarization of macrophages in the moxibustion group changed, and that was manifested by enhanced M2-polarized Mφs and inhibited M1-polarized Mφs. Meanwhile, moxibustion suppressed the activation of JAK1, JAK3 and STAT6 in the IL-4/STAT6 signaling pathway, which contributed to the polarization of M2 . CONCLUSION: The results demonstrate that moxibustion not only suppresses the polarization of M1, but also promotes the polarization of M1. The anti-inflammatory effect of moxibustion may be related to the regulation of macrophage polarization through IL-4/STAT6 signaling pathway.
Assuntos
Artrite Reumatoide , Interleucina-4 , Macrófagos , Moxibustão , Fator de Transcrição STAT6 , Animais , Anti-Inflamatórios , Artrite Reumatoide/genética , Artrite Reumatoide/terapia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Hiperemia , Interleucina-4/metabolismo , Macrófagos/metabolismo , Ratos , Fator de Transcrição STAT6/metabolismoRESUMO
Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Krüppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.
Assuntos
Terapia por Acupuntura , Polaridade Celular/genética , Hemorragia Cerebral/genética , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Microglia/fisiologia , Animais , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , Ratos , Transdução de SinaisRESUMO
In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.
Assuntos
Micronutrientes , Proteoma , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma , Fator de Crescimento Transformador beta1/fisiologia , Zinco/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Células Cultivadas , Impedância Elétrica , Matriz Extracelular/metabolismo , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/prevenção & controle , Microvilosidades/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Pigmentação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/fisiologia , Zinco/metabolismoRESUMO
Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.
Assuntos
Comunicação Celular/genética , Inflamação/genética , Interleucina-1beta/genética , Ativação de Macrófagos/genética , Comunicação Celular/efeitos da radiação , Polaridade Celular/genética , Polaridade Celular/efeitos da radiação , Técnicas de Cocultura , Campos Eletromagnéticos , Humanos , Inflamação/imunologia , Inflamação/terapia , Macrófagos/imunologia , Macrófagos/metabolismo , Magnetoterapia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Transdução de Sinais , Traumatismos dos Tendões/genética , Traumatismos dos Tendões/patologia , Traumatismos dos Tendões/terapia , Tendões/metabolismo , Tendões/patologia , Tendões/efeitos da radiação , Fator de Necrose Tumoral alfa/genética , Cicatrização/genética , Cicatrização/efeitos da radiaçãoRESUMO
Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-derived tissues like brain, kidney and retina. CRB2 has a ubiquitous expression and has been detected in embryonic brain tissue; but currently there is no data regarding its localization in the adult brain. In our study, we characterized the presence of CRB2 in adult mice brain, where it is particularly enriched in cortex, hippocampus, hypothalamus and cerebellum. Double immunofluorescence analysis confirmed that CRB2 is a neuron-specific protein, present in both soma and projections where colocalizes with certain populations of exocytic and endocytic vesicles and with other members of the Crumbs complex. Finally, in the cortex of CRB1rd8 mutant mice that contain a mutation in the Crb1 gene generating a truncated CRB1 protein, there is an abnormal increase in the expression levels of the CRB2 protein which suggests a possible compensatory mechanism for the malfunction of CRB1 in this mutant background.
Assuntos
Encéfalo/metabolismo , Polaridade Celular/genética , Proteínas de Membrana/genética , Neurônios/metabolismo , Animais , Cerebelo/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Hipotálamo/metabolismo , Camundongos , Mutação , Neurônios/patologia , Transdução de Sinais/genéticaRESUMO
Planar cell polarity (PCP) describes a cell-cell communication process through which individual cells coordinate and align within the plane of a tissue. In this study, we show that overexpression of Fuz, a PCP gene, triggers neuronal apoptosis via the dishevelled/Rac1 GTPase/MEKK1/JNK/caspase signalling axis. Consistent with this finding, endogenous Fuz expression is upregulated in models of polyglutamine (polyQ) diseases and in fibroblasts from spinocerebellar ataxia type 3 (SCA3) patients. The disruption of this upregulation mitigates polyQ-induced neurodegeneration in Drosophila We show that the transcriptional regulator Yin Yang 1 (YY1) associates with the Fuz promoter. Overexpression of YY1 promotes the hypermethylation of Fuz promoter, causing transcriptional repression of Fuz Remarkably, YY1 protein is recruited to ATXN3-Q84 aggregates, which reduces the level of functional, soluble YY1, resulting in Fuz transcriptional derepression and induction of neuronal apoptosis. Furthermore, Fuz transcript level is elevated in amyloid beta-peptide, Tau and α-synuclein models, implicating its potential involvement in other neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Taken together, this study unveils a generic Fuz-mediated apoptotic cell death pathway in neurodegenerative disorders.
Assuntos
Apoptose , Polaridade Celular/genética , Polaridade Celular/fisiologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Adulto , Idoso , Peptídeos beta-Amiloides/metabolismo , Animais , Caspase 3/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Proteínas Desgrenhadas/metabolismo , Drosophila , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Doenças Neurodegenerativas/induzido quimicamente , Peptídeos/farmacologia , Ratos , Fator de Transcrição YY1/genética , alfa-Sinucleína/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas tau/metabolismoRESUMO
As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in Arabidopsis pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a cytosolic phosphoglycerate kinase (pgkc-1) mutant with accelerated pollen germination and compromised pollen tube growth polarity. pgkc-1 mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, pgkc-1 pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the pgkc-1 phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the pgkc-1 pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Glicólise , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Padronização Corporal/genética , Padronização Corporal/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Genes de Plantas , Germinação/genética , Germinação/fisiologia , Glicólise/genética , Modelos Biológicos , Mutação , Fosfoglicerato Quinase/antagonistas & inibidores , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Dendropanax morbifera (D. morbifera), a species endemic to Korea, is largely distributed throughout the southern part of the country. Its leaves, stems, roots, and seeds have been used as a form of alternative medicine for various diseases and neurological disorders including paralysis, stroke, and migraine. However, the molecular mechanisms that underlie the remedial effects of D. morbifera remain largely unknown. In this paper, extracts from D. morbifera leaves were prepared using ethyl acetate as a solvent (abbreviated as DMLE). The modulatory effects of DMLE on neuroinflammation were studied in a lipopolysaccharide (LPS)-stimulated BV2 murine microglial cell line. Production of pro-inflammatory cytokines, activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-[Formula: see text]B), and different M1/M2 activation states of microglia were examined. DMLE treatment suppressed the production of pro-inflammatory cytokines including tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]), interleukin-6 (IL-6), and nitric oxide (NO) in LPS-stimulated BV2 cells. DMLE treatment also attenuated the activation of MAPKs and NF-[Formula: see text]B. In a novel discovery, we found that DMLE up-regulated the marker genes representing an alternative, anti-inflammatory M2 polarization, while suppressing the expression of the classical, pro-inflammatory M1 activation state genes. Here, we uncovered the cellular mechanisms underlying the beneficial effects of D. morbifera against neuroinflammation using BV2 microglia cells. These results strongly suggest that DMLE was able to counter the effects of LPS on BV2 cells via control of microglia polarization states. Additionally, study results indicated that DMLE may have therapeutic potential as a neuroinflammation-suppressing treatment for neurodegenerative diseases.
Assuntos
Araliaceae/química , Microglia/patologia , Inflamação Neurogênica/tratamento farmacológico , Fitoterapia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/efeitos adversos , Camundongos , Microglia/metabolismo , Terapia de Alvo Molecular , Doenças Neurodegenerativas/tratamento farmacológico , Inflamação Neurogênica/induzido quimicamente , Inflamação Neurogênica/genética , Óxido Nítrico/metabolismo , Folhas de Planta , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the antiproliferative and anti-metastasis effect of Xihuang Pill (, XP) on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects. METHODS: Highly metastatic human colorectal cancer cell line LoVo was treated with low-, medium-, and highdose XP-containing serum (XP-L, XP-M, XP-H) groups for 48 h, cells intervened with no drug rat serum and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor] as negative and positive controls (NC and PC) groups. Cell proliferation assay was made using cell counting kit-8 (CCK8). The 8 µm pore-size transwell chamber and 4', 6-diamidino-2-phenylindole (DAPI) staining were applied to examine the ability of invasion and migration of the cells. The protein expression of ERK1/2, zinc fifi nger E-box-binding homeobox 1 (ZEB1), Scrib and lethal giant larvae homolog 2 (Lgl2) was detected by Western blotting while the relative mRNA quantity of E-cadherin, N-cadherin, Occludin and junctional adhesion molecule-1 (JAM1) was measured by realtime fluorescent quantitative polymerase chain reaction (RT-qPCR). RESULTS: XP induced a dose-dependent suppression on the proliferation of LoVo cells (P <0.05 or P<0.01), with the inhibition rates varied from 27.30% to 31.08%. Transwell assay showed that when preprocessed with PD98059 and XP-containing serum, the number of cells that passed the filter decreased significantly compared with that of NC group (P <0.05 or P<0.01). Moreover, XP inhibited the protein expression of ERK1/2 and ZEB1 (P <0.05); and up-regulated the protein expression of Scrib and Lgl2 (P <0.05). The mRNA levels of E-cadherin, Occludin and JAM1 of the XP intervened groups and PC group markedly ascended (P <0.05) while that of N-cadherin showed a descending tendency (P>0.05). CONCLUSION: XP intervention suppressed the ability of proliferation, invasion and migration of the LoVo cells. Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.
Assuntos
Polaridade Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Medicamentos de Ervas Chinesas/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Polaridade Celular/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Invasividade Neoplásica , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.
Assuntos
Técnicas de Transferência de Genes , Neurônios/patologia , Canais de Potássio de Domínios Poros em Tandem/genética , Estado Epiléptico/genética , Estado Epiléptico/prevenção & controle , Animais , Morte Celular/genética , Polaridade Celular/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Masculino , Inibição Neural/genética , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/administração & dosagem , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/patologiaRESUMO
Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Polaridade Celular/fisiologia , Endossomos/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Ribosilação do ADP/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridade Celular/genética , Endossomos/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pólen/genética , Pólen/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/genética , Fatores de Transcrição/genéticaRESUMO
T cell immune dysfunction has an important role in the profound immune suppression that characterizes chronic lymphocytic leukemia (CLL). Improper polarization of T cells has been proposed as one of the mechanism involved. Mounting data implicates chromatin regulation, namely promoter methylation, in the plasticity of naïve human T cells. Recent in vitro evidence indicates that this plasticity may be phenotypically altered by using methylation inhibitors which are approved for clinical use in certain types of cancer. These results beg the question: can the ineffective polarization of T lymphocytes in the context of CLL be effectively modulated using methylation inhibitors in a sustainable therapeutic fashion? To answer this question our laboratory has studied the effects of 5-aza-2'-deoxycytidine (5A2) in helper and cytotoxic T lymphocytes from healthy donors and CLL patients in well characterized molecular and epigenetic signaling pathways involved in effective polarization. Moreover, we sought to investigate the consequences of methylation inhibitor treatment on lymphocyte survival, activation intensity, and naïve cell polarization. Our data indicates that 5A2 treatment can depolarize Th2 cells to effectively secrete interferon gamma, signal via T-bet, and achieve demethylation of critical Th1 specific promoters. Moreover, we demonstrate that 5A2 can force Th1 polarization of naïve T cells despite a strong IL-4 stimuli and a lack of IL-12. In conclusion our data seeks to define a modality in which improper or ineffective T cell polarization can be altered by 5AZA and could be incorporated in future therapeutic interventions.
Assuntos
Azacitidina/análogos & derivados , Polaridade Celular/genética , Epigênese Genética/fisiologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Epigênese Genética/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/fisiologiaRESUMO
The epicardium is the last layer of the vertebrate heart to form, surrounding the heart muscle during embryogenesis and providing signaling cues essential to the continued growth and differentiation of the heart. This outer layer of the heart develops from a transient structure, the proepicardial organ (PEO). Despite its essential roles, the early signals required for the formation of the PEO and the epicardium remain poorly understood. The molecular markers wt1 and tcf21 are used to identify the epicardial layer in the zebrafish heart, to trace its development and to determine genes required for its normal development. Disruption of lateral plate mesoderm (LPM) migration through knockdown of miles apart or casanova leads to cardia bifida with each bilateral heart associated with its own PEO, suggesting that the earliest progenitors of the epicardium lie in the LPM. Using a gene knockdown approach, a genetic framework for PEO development is outlined. The pandora/spt6 gene is required for multiple cardiac lineages, the zinc-finger transcription factor wt1 is required for the epicardial lineage only and finally, the cell polarity genes heart and soul and nagie oko are required for proper PEO morphogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Pericárdio/embriologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Biomarcadores , Linhagem da Célula , Polaridade Celular/genética , DNA Complementar , Embrião não Mamífero , Feminino , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Hibridização In Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microinjeções , Modelos Biológicos , Mutação , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Pericárdio/citologia , Pericárdio/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Fatores de Transcrição SOX , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Neural tube defects (NTDs) represent a common group of severe congenital malformations that result from failure of neural tube closure during early development. Their etiology is quite complex involving environmental and genetic factors and their underlying molecular and cellular pathogenic mechanisms remain poorly understood. Animal studies have recently demonstrated an essential role for the planar cell polarity pathway (PCP) in mediating a morphogenetic process called convergent extension during neural tube formation. Alterations in members of this pathway lead to NTDs in vertebrate models, representing novel and exciting candidates for human NTDs. Genetic studies in NTDs have focused mainly on folate-related genes based on the finding that perinatal folic acid supplementation reduces the risk of NTDs by 60-70%. A few variants in these genes have been found to be significantly associated with an increased risk for NTDs. The candidate gene approach investigating genes involved in neurulation has failed to identify major causative genes in the etiology of NTDs. Despite this history of generally negative findings, we are achieving a rapid and impressive progress in understanding the genetic basis of NTDs, based mainly on the powerful tool of animal models.
Assuntos
Defeitos do Tubo Neural/genética , Animais , Divisão Celular/genética , Polaridade Celular/genética , Sistema Nervoso Central/embriologia , Cílios/fisiologia , Cílios/ultraestrutura , Modelos Animais de Doenças , Feminino , Ácido Fólico/metabolismo , Humanos , Recém-Nascido , Camundongos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/etiologia , Defeitos do Tubo Neural/metabolismo , Gravidez , Transdução de Sinais/genéticaRESUMO
Rhotekin, an effector of Rho, is highly expressed in the brain but its function(s) in neurons is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin by yeast two-hybrid screening of human brain cDNA library. We then found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in COS7 cells and the rat brain. The C-terminal three amino acids of Rhotekin were essential for the interaction with Lin-7B. Their binding affinity became increased in the presence of active RhoA in the COS7 cell expression system. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.
Assuntos
Polaridade Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Química Encefálica , Células COS , Células Cultivadas , Chlorocebus aethiops , Giro Denteado/citologia , Giro Denteado/metabolismo , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Proteínas de Ligação ao GTP , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Plasmídeos/genética , Ligação Proteica , Tálamo/citologia , Tálamo/metabolismo , TransfecçãoRESUMO
Vesicle trafficking is essential for the generation of asymmetries, which are central to multicellular development. Core components of the vesicle transport machinery, such as ADP-ribosylation factor (ARF) GTPases, have been studied primarily at the single-cell level. Here, we analyze developmental functions of the ARF1 subclass of the Arabidopsis thaliana multigene ARF family. Six virtually identical ARF1 genes are ubiquitously expressed, and single loss-of-function mutants in these genes reveal no obvious developmental phenotypes. Fluorescence colocalization studies reveal that ARF1 is localized to the Golgi apparatus and endocytic organelles in both onion (Allium cepa) and Arabidopsis cells. Apical-basal polarity of epidermal cells, reflected by the position of root hair outgrowth, is affected when ARF1 mutants are expressed at early stages of cell differentiation but after they exit mitosis. Genetic interactions during root hair tip growth and localization suggest that the ROP2 protein is a target of ARF1 action, but its localization is slowly affected upon ARF1 manipulation when compared with that of Golgi and endocytic markers. Localization of a second potential target of ARF1 action, PIN2, is also affected with slow kinetics. Although extreme redundancy precludes conventional genetic dissection of ARF1 functions, our approach separates different ARF1 downstream networks involved in local and specific aspects of cell polarity.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Epiderme Vegetal/citologia , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Polaridade Celular/genética , Proteínas de Ligação a DNA/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Família Multigênica , Cebolas/genética , Fenótipo , Epiderme Vegetal/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.