Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.Mitochondria perform numerous essential physiological functions in all eukaryotic cells. Apart from their role in oxidative phosphorylation and fatty acid oxidation, they are also essential for biosynthesis of central building blocks such as amino acids and nucleotides. At the same time, mitochondria are a threat to cellular well-being. Mitochondria are a major source of reactive oxygen species in cells. In addition, disruption of mitochondrial compartmentalization results in leakage of cytochrome c and other cytotoxic factors, and mitochondria with defective chemiosmotic coupling can cause an energy drain on the cell. Accumulation of mitochondrial genetic variation and mitochondrial damage are widely considered to underlie many age-related metabolic diseases and late-onset genetic disorders (1, 2). It is commonly postulated that in normal cells defective mitochondria are broken down in the lysosomal compartment through autophagy, and inability to clear defective mitochondria is thought to underlie numerous pathological conditions (3, 4).Autophagy is a set of catabolic membrane trafficking mechanisms that allow import of cytosolic material into the vacuole/ lysosome. The best understood form of autophagy is macroautophagy, in which intracellular membranes of undetermined origin engulf cytosolic material to form a double or multi-bilayer membrane bound intermediate called the autophagosome (reviewed in Refs. 3 and 5-8). This intermediate then goes on to fuse with the vacuole/lysosome, releasing a single-bilayer bound vesicle called an autophagic body into the lumen of the lytic compartment where it is broken down, releasing the cytosol-derived material for further degradation to biosynthetic building blocks. Cl...
SUMMARYLidocaine and related local anaesthetics have been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms underlying their therapeutic effect are poorly defined. Intestinal epithelial cells play an important role in the mucosal inflammatory response that leads to tissue damage in UC via the secretion of pro-inflammatory cytokines and chemokines. The aim of this study was to evaluate the direct immunoregulatory effect of lidocaine on pro-inflammatory cytokine and chemokine secretion from intestinal epithelial cells. HT-29 and Caco-2 cell lines were used as a model system and treated with lidocaine and related drugs. The expression of IL-8, IL-1b and the IL-1 receptor antagonist (RA) were assessed by ELISA and quantification of mRNA. In further experiments, the effect of lidocaine on the secretion of IL-8 from freshly isolated epithelial cells stimulated with TNFa was tested. Lidocaine, in therapeutic concentrations, inhibited the spontaneous and TNFa-stimulated secretion of IL-8 and IL-1b from HT-29 and Caco-2 cell lines in a dose-dependent manner. Similarly, suppression of IL-8 secretion was noted in the freshly isolated epithelial cells. Other local anaesthetics, bupivacaine and amethocaine, had comparable effects. Lidocaine stimulated the secretion of the anti-inflammatory molecule IL-1 RA. Both the inhibitory and the stimulatory effects of lidocaine involved regulation of transcription. The results imply that the therapeutic effect of lidocaine may be mediated, at least in part, by its direct effects on epithelial cells to inhibit the secretion of proinflammatory molecules on one hand while triggering the secretion of anti-inflammatory mediators on the other.
Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-α- and Salmonella-induced secretion of IL-8 and IL-1β derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-α-induced secretion of IL-8 and IL-1β. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1β after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1β similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1β. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.
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