RESUMO
The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process.
Assuntos
Fagocitose , Epitélio Pigmentado da Retina , Antígenos CD36/genética , Antígenos CD36/metabolismo , Lisossomos/metabolismo , Fagocitose/genética , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Corticotrofos/metabolismo , Degradação Associada com o Retículo Endoplasmático , Hexosiltransferases/genética , Proteínas de Membrana/genética , Selenoproteínas/genética , Transdução de Sinais , Hormônio Adrenocorticotrópico/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Corticotrofos/citologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Glicosilação , Hexosiltransferases/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Microssomos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno , Selenoproteínas/antagonistas & inibidores , Selenoproteínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Pre-mRNA splicing is a critical step in RNA processing in all eukaryotic cells. It consists of introns removal and requires the assembly of a large RNA-protein complex called the spliceosome. This complex of small nuclear ribonucleoproteins is associated with accessory proteins from the pre-mRNA processing factor (PRPF) family. Mutations in different splicing factor-encoding genes were identified in retinitis pigmentosa (RP) patients. A surprising feature of these ubiquitous factors is that the outcome of their alteration is restricted to the retina. Because of their high metabolic demand, most studies focused on photoreceptors dysfunction and associated degeneration. However, cells from the retinal pigment epithelium (RPE) are also crucial to maintaining retinal homeostasis and photoreceptor function. Moreover, mutations in RPE-specific genes are associated with some RP cases. Indeed, we identified major RPE defects in Prpf31-mutant mice: circadian rhythms of both photoreceptor outer segments (POS) phagocytosis and retinal adhesion were attenuated or lost, leading to ultrastructural anomalies and vacuoles. Taken together, our published and ongoing data suggest that (1) similar molecular events take place in human and mouse cells and (2) these functional defects generate various stress processes.
Assuntos
Células Epiteliais/patologia , Proteínas do Olho/genética , Retinose Pigmentar/genética , Animais , Ritmo Circadiano , Células Epiteliais/ultraestrutura , Humanos , Camundongos , Fagocitose , Células Fotorreceptoras de Vertebrados/patologia , Fatores de Processamento de RNA/genética , Epitélio Pigmentado da Retina/citologia , Retinose Pigmentar/patologiaRESUMO
We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that ß-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of ß-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.
Assuntos
Ciclina A2/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fosfolipases Tipo C/metabolismo , beta Catenina/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclina A2/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Fosfolipases Tipo C/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Several cues including pituitary adenylate cyclase-activating polypeptide (PACAP), which acts through cAMP stimulation, specify the conversion of sympathoadrenal (SA) precursors toward different cell phenotypes by promoting their survival and differentiation. Selenoprotein T (SELENOT) is a PACAP-stimulated ER oxidoreductase that exerts an essential antioxidant activity and whose up-regulation is associated with SA cell differentiation. In the present study, we investigated the transcriptional cascade elicited by PACAP/cAMP to trigger SELENOT gene transcription during the conversion of PC12 cells from SA progenitor-like cells toward a neuroendocrine phenotype. Unexpectedly, we found that PACAP/cAMP recruits the canonical pathway that regulates mitochondrial function in order to elicit SELENOT gene transcription and the consequent antioxidant response during PC12 cell differentiation. This cascade involves LKB1-mediated AMPK activation in order to stimulate SELENOT gene transcription through the PGC1-α/NRF-1 complex, thus allowing SELENOT to promote PACAP-stimulated neuroendocrine cell survival and differentiation. Our data reveal that a PACAP and cAMP-activated AMPK-PGC-1α/NRF-1 cascade is critical for the coupling of oxidative stress tolerance, via SELENOT gene expression, and mitochondrial biogenesis in order to achieve PC12 cell differentiation. The data further highlight the essential role of SELENOT in cell metabolism during differentiation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Neuroendócrinas/citologia , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Selenoproteínas/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Modelos Biológicos , Células Neuroendócrinas/efeitos dos fármacos , Células Neuroendócrinas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ligação Proteica/efeitos dos fármacos , Ratos , Selenoproteínas/metabolismoRESUMO
AIMS: Oxidative stress is central to the pathogenesis of Parkinson's disease (PD), but the mechanisms involved in the control of this stress in dopaminergic cells are not fully understood. There is increasing evidence that selenoproteins play a central role in the control of redox homeostasis and cell defense, but the precise contribution of members of this family of proteins during the course of neurodegenerative diseases is still elusive. RESULTS: We demonstrated first that selenoprotein T (SelT) whose gene disruption is lethal during embryogenesis, exerts a potent oxidoreductase activity. In the SH-SY5Y cell model of dopaminergic neurons, both silencing and overexpression of SelT affected oxidative stress and cell survival. Treatment with PD-inducing neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone triggered SelT expression in the nigrostriatal pathway of wild-type mice, but provoked rapid and severe parkinsonian-like motor defects in conditional brain SelT-deficient mice. This motor impairment was associated with marked oxidative stress and neurodegeneration and decreased tyrosine hydroxylase activity and dopamine levels in the nigrostriatal system. Finally, in PD patients, we report that SelT is tremendously increased in the caudate putamen tissue. INNOVATION: These results reveal the activity of a novel selenoprotein enzyme that protects dopaminergic neurons against oxidative stress and prevents early and severe movement impairment in animal models of PD. CONCLUSIONS: Our findings indicate that selenoproteins such as SelT play a crucial role in the protection of dopaminergic neurons against oxidative stress and cell death, providing insight into the molecular underpinnings of this stress in PD.