The role of intracellular labile iron in redox signal transductions (Doctoral thesis)

Ρουπάκα, Βασιλική

Iron (Fe) is an essential element for a variety of important biological processes. The ability of iron to exist in various oxidation states makes it an essential element in many cellular processes associated with basic physiological cell functions. On the other hand, the same high redox potential that enables iron to readily switch between the ferrous and ferric states also makes it potentially toxic. Although, iron is usually bound with high affinity to specialized proteins in order to abrogate the deleterious effects of free iron during oxidative stress, a distinct minor fraction of total cellular iron is thought to be loosely bound to diverse intracellular ligants and serves as a crossroad of cell iron metabolism. This fraction of iron is called redox-active iron and in the presence of H2O2, it catalyzes the generation of the extremely reactive hydroxyl radical (HO·), which rapidly react and oxidize the cellular components. Recent studies of our research group have shown that redox active iron appears to be essential for the signal transduction. Redox active iron is specifically responsible for the sustained activation of the MAP kinase JNK/p38 axis, which is critical for apoptotic cell death. In this study, we further investigated the role of redox active iron in H2O2 induced signaling. Furthermore, we investigated the role of Trxl and Grxl on the phosphorylation of the three MAP kinases (JNK, p38, ERK) phosphorylation under oxidative stress conditions. For this purpose, we preincubated HI299 cell line (human pulmonary cancer cells) with a specific iron chelator desferioxamine (DFO) and then challenged with H2O2. DFO represents a strong iron chelator, which is taken up by fluid phase endocytosis, reaching the lysosomal compartment. Iron depletion by DFO attenuated the second prolonged phases of JNK and p38, but had slight effect on ERK phosphorylation, which is regulated cell survival and proliferation. Next we tested the role of Trxl and Grxl, which involved in maintenance of intracellular redox homeostasis, on MAP kinases phosphorylation. For this purpose, Trxl and Grxl were stably overexpressed or silenced in the same HI299 cell line. Upon H2O2 treatment, overexpression of Trxl reduced the sustained phosphorylation of JNK and the two phosphorylation phases of p38. On the other hand, overexpression of Grxl reduced the sustained phosphorylation of JNK and mainly the sustained phosporylation of p38. In addition, downregulation of Trxl promoted the sustained phosphorylation of JNK and the two phosphorylation phases of p38 upon H2O2 challenge. On the contrary, downregulation of Grxl increased mainly the prolonged phases of JNK and p38 phosphorylation. Interestingly, overexpression/silencing of Trxl and Grxl respectively, had no effect on ERK phosphorylation. The phosphorylation levels of MAP kinases depend on upstream kinases MAPKK and MAPKKK. It is proposed that ASK1, a special MAPKKK, is responsible for the phosphorylation of JNK and p38. After cell exposure to H2O2, it was observed two phases of disulfide bond formation of this kinase. The oxidation of cysteine residues in ASK1 resulting at the formation of disulfide bonds among ASK1 molecules or other proteins. Afterwards, the role of labile iron in H2O2- induced oxidation of ASK1 was investigated. Interestingly, the second phase of ASK1 oxidation was attenuated by DFO, implicating labile iron in ASK1 oxidation.In conclusion intracellular labile iron, Trxl and Grxl have a more specific role in H2O2 induced apoptosis. However, the precise role of these molecules has to be elucidated.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής
Subject classification: Σίδηρος
Keywords: Καταλυτικά ενεργός σίδηρος,Δεσφεριοξαμίνη,Υπεροξείδιο του υδρογόνου,Απόπτωση,Θειορεδοξίνη 1,Γλουταρεδοξίνη 1,Labile Iron Pool (LIP),Desferrioxamine,Hydrogen peroxide,Apoptosis,Thioredoxin 1,Glutaredoxin 1
Appears in Collections:Διδακτορικές Διατριβές

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