RESUMO
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Transforming growth factor beta (TGF-ß) is highly expressed in the liver tumor microenvironment and is known to inhibit immune cell activity. Here, we used human induced pluripotent stem cells (iPSCs) to produce natural killer (NK) cells engineered to mediate improved anti-HCC activity. Specifically, we produced iPSC-NK cells with either knockout TGF-ß receptor 2 (TGFBR2-KO) or expression of a dominant negative (DN) form of the TGF-ß receptor 2 (TGFBR2-DN) combined with chimeric antigen receptors (CARs) that target either GPC3 or AFP. The TGFBR2-KO and TGFBR2-DN iPSC-NK cells are resistant to TGF-ß inhibition and improved anti-HCC activity. However, expression of anti-HCC CARs on iPSC-NK cells did not lead to effective anti-HCC activity unless there was also inhibition of TGF-ß activity. Our findings demonstrate that TGF-ß signaling blockade is required for effective NK cell function against HCC and potentially other malignancies that express high levels of TGF-ß.
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Protease activated receptors (PARs) are among the first receptors shown to transactivate other receptors: noticeably, these interactions are not limited to members of the same family, but involve receptors as diverse as receptor kinases, prostanoid receptors, purinergic receptors and ionic channels among others. In this review, we will focus on the evidence for PAR interactions with members of their own family, as well as with other types of receptors. We will discuss recent evidence as well as what we consider as emerging areas to explore; from the signalling pathways triggered, to the physiological and pathological relevance of these interactions, since this additional level of molecular cross-talk between receptors and signaling pathways is only beginning to be explored and represents a novel mechanism providing diversity to receptor function and play important roles in physiology and disease.
Assuntos
Receptores Ativados por Proteinase , Transdução de Sinais , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais/fisiologiaRESUMO
AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.
Assuntos
Bestrofinas/metabolismo , Ácido Glutâmico/metabolismo , Peptídeos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Peptídeos/administração & dosagem , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/citologiaRESUMO
PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Receptor PAR-1/metabolismo , Retina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Humanos , Retina/citologiaRESUMO
Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.
Assuntos
Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/citologia , Trocador de Sódio e Cálcio/metabolismo , Trombina/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Forma Celular/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/metabolismo , Fragmentos de Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos Long-Evans , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trítio/metabolismoRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.
Assuntos
Ataxina-7/metabolismo , Proteínas Mutantes/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ataxias Espinocerebelares/fisiopatologia , Visão Ocular/efeitos dos fármacos , Animais , Ataxina-7/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Peptídeos/metabolismo , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Agregados Proteicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/complicações , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/patologiaRESUMO
BACKGROUND AND OBJECTIVE: To demonstrate the advantage of optical coherence tomography angiography (OCTA) for the diagnosis and management of proliferative macular telangiectasia type 2 (MacTel2) masquerading as neovascular age-related macular degeneration (AMD). PATIENTS AND METHODS: This is an observational cases series. Three patients referred with the diagnosis of neovascular AMD were identified in this retrospective study. In addition to color fundus, fluorescein angiography, and spectral-domain OCT (SD-OCT) imaging, SD-OCTA (AngioPlex; Carl Zeiss Meditec, Dublin, CA) was performed. RESULTS: SD-OCTA revealed bilateral parafoveal retinal microvascular changes in three patients and unambiguously confirmed the diagnosis of MacTel2. CONCLUSION: OCTA is an important tool for the correct diagnosis of MacTel2 in older patients with the concomitant or masquerading diagnosis of AMD. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:303-312.].
Assuntos
Neovascularização de Coroide/diagnóstico , Angiografia por Tomografia Computadorizada , Técnicas de Diagnóstico Oftalmológico , Degeneração Macular/diagnóstico , Telangiectasia Retiniana/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by Gq11α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of µ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel µ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.
RESUMO
Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK)-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis.
Assuntos
Movimento Celular , Paxilina/metabolismo , Animais , Adesões Focais/metabolismo , Humanos , Patologia Molecular , Fosforilação , Transdução de SinaisRESUMO
PURPOSE: To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process. METHODS: Protease-activated receptor (PARs 1-4) expression was analyzed using RT-PCR and Western blot in the MIO-M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal-regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition. RESULTS: We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ. CONCLUSIONS: The breakdown of blood-retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.
Assuntos
Células Ependimogliais/enzimologia , Hemostáticos/farmacologia , Proteína Quinase C/fisiologia , Trombina/farmacologia , Análise de Variância , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Ependimogliais/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Ativados por Proteinase/metabolismo , Vitreorretinopatia Proliferativa/enzimologia , Vitreorretinopatia Proliferativa/etiologia , Cicatrização/fisiologiaRESUMO
Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function.
Assuntos
Células Ependimogliais/metabolismo , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Receptores de Glutamato/metabolismo , Regulação para Cima/fisiologia , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Células Cultivadas , Células Ependimogliais/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Neuroglia/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
The breakdown of the blood-retina barrier exposes retinal pigment epithelium (RPE) to serum components, thrombin among them. In addition to coagulation, thrombin acting through Protease-Activated Receptors (PARs 1-4) participates in a number of processes including cell proliferation, transformation, and migration. The purpose of this study was to identify interacting signaling pathways by which the activation of PAR1 by thrombin triggers cyclin D1 gene (Ccnd1) expression and the proliferation of RPE cells, characteristic of proliferative vitreoretinopathy (PVR). Our results demonstrate that thrombin induces the expression of the c-fos gene (c-fos), the activation of the (fos/jun) AP-1 site and the expression of Ccnd1, in precise correlation with the activation of CREB. Although the expression of both, c-fos and Ccnd1 requires the activation of conventional PKC isoforms and PI3K, downstream signaling from PI3K differs for both genes. Whereas the expression of c-fos requires PI3K-induced PDK1/Akt activity, that of Ccnd1 is mediated by PDK1-independent PKCζ signaling. Additionally, CREB activation may contribute to the induction of Ccnd1 expression through binding to the Ca/CRE element in the Ccnd1 gene promoter. Since cyclin D1 is a key regulator of cell cycle G1/S phase progression essential for proliferation, these findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.
Assuntos
Ciclina D1/genética , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Vitreorretinopatia Proliferativa/genética , Barreira Hematorretiniana/efeitos dos fármacos , Western Blotting , Proliferação de Células , Células Cultivadas , Ciclina D1/biossíntese , Hemostáticos/farmacologia , Humanos , Reação em Cadeia da Polimerase , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Trombina/farmacologia , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismoRESUMO
X-linked spinal and bulbar muscular atrophy (SBMA) is characterized by adult-onset muscle weakness and lower motor neuron degeneration. SBMA is caused by CAG-polyglutamine (polyQ) repeat expansions in the androgen receptor (AR) gene. Pathological findings include motor neuron loss, with polyQ-AR accumulation in intranuclear inclusions. SBMA patients exhibit myopathic features, suggesting a role for muscle in disease pathogenesis. To determine the contribution of muscle, we developed a BAC mouse model featuring a floxed first exon to permit cell-type-specific excision of human AR121Q. BAC fxAR121 mice develop systemic and neuromuscular phenotypes, including shortened survival. After validating termination of AR121 expression and full rescue with ubiquitous Cre, we crossed BAC fxAR121 mice with Human Skeletal Actin-Cre mice. Muscle-specific excision prevented weight loss, motor phenotypes, muscle pathology, and motor neuronopathy and dramatically extended survival. Our results reveal a crucial role for muscle expression of polyQ-AR in SBMA and suggest muscle-directed therapies as effective treatments.
Assuntos
Encéfalo/patologia , Músculo Esquelético/metabolismo , Transtornos Musculares Atróficos/genética , Transtornos Musculares Atróficos/patologia , Peptídeos/genética , Receptores Androgênicos/genética , Actinas/genética , Actinas/metabolismo , Fatores Etários , Animais , Peso Corporal/genética , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Força Muscular/genética , Músculo Esquelético/patologia , Transtornos Musculares Atróficos/complicações , Fenótipo , Receptores Androgênicos/metabolismoRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.
Assuntos
Alelos , Doença de Huntington/genética , Doença de Huntington/patologia , Mutação/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Ansiedade/genética , Ansiedade/patologia , Ansiedade/fisiopatologia , Sistema Nervoso Autônomo/patologia , Sistema Nervoso Autônomo/fisiopatologia , Comportamento Animal , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Técnicas de Introdução de Genes , Glucose/metabolismo , Proteína Huntingtina , Doença de Huntington/fisiopatologia , Aprendizagem , Pulmão/anormalidades , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Atividade Motora , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Fenótipo , Teste de Desempenho do Rota-Rod , Olfato , Comportamento SocialRESUMO
The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSß (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Doença de Huntington/genética , Proteínas Nucleares/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Modelos Animais de Doenças , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Humanos , Doença de Huntington/patologia , Camundongos , Proteína 1 Homóloga a MutL , Proteínas MutL , RNA MensageiroRESUMO
Epithelial-mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-ζ/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Trombina/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , RNA Interferente Pequeno , Proteína Companheira de mTOR Insensível à Rapamicina , Ratos , Proteína Regulatória Associada a mTOR , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-ß-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Células Ependimogliais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Ácido Aspártico/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Ácido D-Aspártico/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Células Ependimogliais/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Retina/citologia , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.
Assuntos
Modelos Animais de Doenças , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Fenótipo , Degeneração Retiniana/patologia , Análise de Variância , Animais , Temperatura Corporal , Encéfalo/patologia , Eletrorretinografia , Comportamento Exploratório/fisiologia , Feminino , Ferritinas/sangue , Genótipo , Coração/crescimento & desenvolvimento , Imuno-Histoquímica , Linfócitos/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/complicações , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Tamanho do Órgão , Consumo de Oxigênio/fisiologia , Degeneração Retiniana/etiologiaRESUMO
Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.
Assuntos
Hemostáticos/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fibras de Estresse/metabolismo , Trombina/farmacologia , Actinas/metabolismo , Animais , Barreira Hematorretiniana , Western Blotting , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Proteínas Musculares , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-ß inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC-ß-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.