RESUMO
The constitutive secretion of antioxidant Cu-Zn Superoxide dismutase (SOD1) has been widely demonstrated in many cellular lines. In addition, we showed that as well as the basal SOD1 secretion, this enzyme is also exported through depolarization of excitable cells by high extracellular K concentration. Recent data showed that SOD1 was able to activate muscarinic M1 receptor producing the activation, via phospholipase C, of ERK1-2 and AKT pathways. It is also known that about 20% of familial amyotrophic lateral sclerosis (fALS) is due to mutations in the gene coding for SOD1. The aim of the present research is to evaluate whether, analogously to wild type SOD1 (SOD1wt), the mutated form of SOD1G93A is able to activate ERK1-2 and AKT through muscarinic M1 receptor in SK-N-BE as well as in motoneuron like NSC-34. Our results demonstrated that in NSC-34 and SK-N-BE cells mutated SOD1G93A carried out a more evident activation of ERK1-2 and AKT and a stronger increase of intracellular calcium levels compared to SOD1WT; we also demonstrated that these effects are mediated by the M1 receptor as shown using pirenzepine, a specific M1 inhibitor and the calcium chelator BAPTA. Of note, M1 receptor pathway activation by SOD1G93A, but not by SOD1WT, is associated with both an increase of reactive oxygen species and a cytotoxic effect.
RESUMO
Reactive oxygen species (ROS) are signaling molecules that mediate stress response, apoptosis, DNA damage, gene expression and differentiation. We report here that differentiation of oligodendrocytes (OLs), the myelin forming cells in the CNS, is driven by ROS. To dissect the OL differentiation pathway, we used the cell line MO3-13, which display the molecular and cellular features of OL precursors. These cells exposed 1-4 days to low levels of H2O2 or to the protein kinase C (PKC) activator, phorbol-12-Myristate-13-Acetate (PMA) increased the expression of specific OL differentiation markers: the specific nuclear factor Olig-2, and Myelin Basic Protein (MBP), which was processed and accumulated selectively in membranes. The induction of differentiation genes was associated with the activation of ERK1-2 and phosphorylation of the nuclear cAMP responsive element binding protein 1 (CREB). PKC mediates ROS-induced differentiation because PKC depletion or bis-indolyl-maleimide (BIM), a PKC inhibitor, reversed the induction of differentiation markers by H2O2. H2O2 and PMA increased the expression of membrane-bound NADPH oxidases, NOX3 and NOX5. Selective depletion of these proteins inhibited differentiation induced by PMA. Furthermore, NOX5 silencing down regulated NOX3 mRNA levels, suggesting that ROS produced by NOX5 up-regulate NOX3 expression. These data unravel an elaborate network of ROS-generating enzymes (NOX5 to NOX3) activated by PKC and necessary for differentiation of OLs. Furthermore, NOX3 and NOX5, as inducers of OL differentiation, represent novel targets for therapies of demyelinating diseases, including multiple sclerosis, associated with impairment of OL differentiation.
RESUMO
Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2-protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2-protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.
