RESUMO
The unfolded protein response (UPR) is a largely cytoprotective signaling cascade that acts to re-establish homeostasis of the endoplasmic reticulum (ER) under conditions of stress by inducing an early and transient block in general protein synthesis and by increasing the folding and degradative capacity of the cell through an extensive transcriptional program. It is well established that the mechanism for the early translational attenuation during ER stress occurs through phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by activated PERK. Our data demonstrate that when eIF2α is dephosphorylated translation is not fully restored to pre-stressed levels. We found that this correlates with reduced mTOR activity and as a result decreases phosphorylation of 4E-BP1, which negatively regulates assembly of the eIF4F complex and cap-dependent translation. The decrease in mTOR activity and 4E-BP1 phosphorylation is associated with activation of AMP kinase, a negative regulator of mTOR, and in the case of some stress conditions, downregulation of signaling through key components of the PI3K pathway. Furthermore, we show that there is a subset of mRNAs that does not recover from UPR-induced translational repression, including those whose translation is particularly sensitive to loss of eIF4F, such as cyclin D1, Bcl-2 and MMP-9. Together these data implicate reduced mTOR activity and 4E-BP1 hypophosphorylation as a second, more restricted mechanism of translational control occurring somewhat later in the UPR.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Resposta a Proteínas não Dobradas/genética , Retículo Endoplasmático , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Transdução de Sinais , Serina-Treonina Quinases TOR/genéticaRESUMO
Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic ß-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 ß-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic ß-cells.
Assuntos
Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Ácido Palmítico/metabolismo , Esfingolipídeos/metabolismo , Estresse Fisiológico , Animais , Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucosilceramidas/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Metabolismo dos Lipídeos , Metabolômica/métodos , Camundongos , Ácido Palmítico/efeitos adversos , Fenilbutiratos/farmacologia , Biossíntese de Proteínas , Transporte Proteico , Serina C-Palmitoiltransferase/antagonistas & inibidores , Estresse Fisiológico/efeitos dos fármacos , Tapsigargina/toxicidade , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tunicamicina/toxicidadeRESUMO
Displacement of uterine epithelial cells is an important aspect of implantation in the rat and other species, allowing invasion of the blastocyst into the endometrial stroma. Desmosomes, which are part of the lateral junctional complex, function in cell-to-cell adhesion, and are therefore likely to be involved in displacement of uterine epithelial cells at the time of implantation. This study used transmission electron microscopy to study rat uterine epithelial cells during the peri-implantation period to investigate the change in the number of structural desmosomes along the lateral plasma membrane of uterine epithelial cells. We found a significant decrease in the number of desmosomes along the entire lateral plasma membrane as pregnancy progressed. Furthermore, there were also significant decreases in the number of desmosomes on the apical portion of the lateral plasma membrane between all days of pregnancy examined. In addition, on day 6 of pregnancy, the time of attachment, desmosomes were larger and seen as "giant desmosomes." For the first time, this study has shown that there is a significant reduction in cell height and actual number of ultrastructurally observable desmosomes at the time of implantation in the rat. It is proposed that this reduction in desmosome number leads to a decrease in lateral adhesion between uterine epithelial cells at the time of implantation, and hence is involved in the loss of uterine epithelial cells to facilitate blastocyst invasion.
Assuntos
Desmossomos/ultraestrutura , Implantação do Embrião , Células Epiteliais/ultraestrutura , Útero/ultraestrutura , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Desmossomos/fisiologia , Células Epiteliais/fisiologia , Feminino , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica de Transmissão , Gravidez , Ratos , Ratos Wistar , Útero/fisiologiaRESUMO
Uterine epithelium undergoes dramatic changes during early pregnancy in preparation for implantation. We have studied distribution patterns of the desmosomal marker, desmoglein 1&2, in rat uterine epithelial cells during early pregnancy as well as in hormonally stimulated ovariectomised animals. On day 1 of pregnancy as well as in oestradiol treated rats, desmoglein 1&2 staining was localized along the entire length of the lateral plasma membrane. By day 3 and on subsequent days of pregnancy as well as in ovariectomised animals treated with progesterone alone or in combination with oestradiol, desmoglein 1&2 staining was concentrated at the apical portion of the lateral plasma membrane. We suggest that the reorganisation of these desmosomal cadherins is an important component of uterine epithelial receptivity and this relocation is under the control of the ovarian hormone progesterone.