15-Deoxy-⌬12-14 -prostaglandin J 2 (dPGJ2) and thiazolidinediones are known as ligands for the peroxisome proliferator activator receptor ␥ (PPAR␥) a member of the nuclear receptor superfamily. Herein, we show that dPGJ2 activates, in cultured primary astrocytes, Erk, Jnk, p38 MAP kinase, and ASK1, a MAP kinase kinase kinase, which can be involved in the activation of Jnk and p38 MAP kinase. The activation kinetic is similar for the three MAP kinase. The activation of the MAP kinases is detectable around 0.5 h. The activation increases with dPGJ2 in a dose dependent manner (0 -15 M). A scavenger of reactive oxygenated species (ROS), N-acetylcysteine (NAC) at 20 mM, completely suppresses the activation of MAP kinases and ASK1, suggesting a role for oxidative stress in the activation mechanism. Other prostaglandin cyclopentenones than dPGJ2, A 2 , and to a lesser degree, A 1 also stimulate the MAP kinases, although they do not bind to PPAR␥. Ciglitazone (20 M), a thiazolidinedione that mimics several effects of dPGJ2 in different cell types, also activates the three MAP kinase families and ASK1 in cultured astrocytes. However the activation is more rapid (it is detectable at 0.25 h) and more sustained (it is still strong after 4 h). NAC prevents the activation of the three MAP kinase families by ciglitazone. Another thiazolidinedione that binds to PPAR␥, rosiglitazone, does not activate MAP kinases, indicating that the effect of ciglitazone on MAP kinases is independent of PPAR ␥. Ciglitazone and less strongly dPGJ2 activate Erk in undifferentiated cells of the adipocyte cell line 1B8. Ciglitazone also activates Jnk and p38 MAP kinase in these preadipocytes. Our findings suggest that a part of the biological effects of dPGJ2 and ciglitazone involve the activation of the three MAP kinase families probably through PPAR␥-independent mechanisms involving ROS. 15-Deoxy-⌬12-14 -prostaglandinJ2 (dPGJ2) 1 has been shown to bind and to activate peroxisome proliferator-activated receptor ␥ (PPAR␥). This receptor, a member of the ligand-regulated nuclear receptor family, is a molecular target of the thiazolidinedione class of antidiabetic drugs (1, 2), including ciglitazone, pioglitazone, and rosiglitazone. This receptor has a critical role in adipogenesis, glucose metabolism, placental function, and macrophage functions (3-5). It has been suggested that much of the effects of dPGJ2 and thiazolidinediones may be mediated by this receptor. However, Rossi et al. (6) have shown that dPGJ2 also can directly inhibit the IKK subunit of IKK, the protein kinase responsible for the activation of NFB by proinflammatory stimuli. This effect of dPGJ2 can be mimicked by other prostaglandin cyclopentenones and is clearly independent of PPAR␥. dPGJ2 also activates Erk cascade in mesangial cells in a manner probably independent of PPAR␥ as ciglitazone does not mimic this effect (7).In the cellular model studied by our group, rat cultured astrocytes, which express PPAR␥, we observed (this paper) 2 that dPGJ2 promotes apoptosis and prevents...
Chronic treatments with antidepressants active on major depressive disorders influence pathways involved in cell survival and plasticity. As astrocytes seem to play a key role in the protection of brain cells, we investigated in these cells the rapid effects of the antidepressant fluoxetine (Prozac) on signaling cascades and gene induction, which probably play a role in neuroprotection. We show here that fluoxetine alone activates the extracellular signal-regulated-protein kinase (Erk) and p38 mitogen-associated protein (MAP) kinase cascades. RT-PCR revealed that genes, modulated in brain by long-term fluoxetine treatment, are rapidly induced by fluoxetine in cultured astrocytes: brain-derived nerve factor (BDNF) and its receptors, glial-derived nerve factor (GDNF) and deiodinase 3 (D3). Induction of D3 by fluoxetine is inhibited by U0126 and SB203580, suggesting that Erk and p38 MAP kinases are involved. Glial-derived nerve factor (GDNF) induction by fluoxetine is prevented by U0126, suggesting that Erk is implicated. Brain-derived nerve factor (BDNF) induction seems mediated by other signaling pathways. In conclusion, we show that fluoxetine alone rapidly activates mitogen activated protein (MAP) kinase cascades in rat astrocytes and that genes involved in neuroprotection are induced in a few hours in a MAP kinase-dependent or -independent manner.
The iodothyronine deiodinases are a family of selenoproteins that metabolize thyroxine and other thyroid hormones to active and inactive metabolites in a number of tissues including brain. Using primary cultures of rat astroglial cells as a model system, we demonstrate that the mRNA for the type II iodothyronine deiodinase (DII) selenoenzyme is rapidly and markedly induced by forskolin and 8-bromo-cAMP. The induction of DII activity, however, was significantly impaired by culturing cells in selenium-deficient medium for 7 days. Under such conditions, the addition of selenium resulted in a rapid increase in cAMP-induced DII activity that was dose-dependent, with maximal effects noted within 2 h. Cycloheximide blocked this effect of selenium on restoring cAMP-induced DII activity, whereas actinomycin D did not. These data demonstrate that the DII selenoenzyme is expressed in cultured astrocytes and that the induction of DII activity by cAMP analogues appears to be mediated, at least in part, by pretranslational mechanisms. Furthermore, selenium deprivation impairs the expression of DII activity at the level of translation.Recent molecular cloning studies have identified cDNAs that code for a family of structurally related iodothyronine deiodinases (1-4). These oxidoreductases catalyze the removal of iodide from the phenolic (5Ј-position) or the tyrosyl (5-position) ring of thyroxine (T 4 ) 1 to form the active compound 3,5,3Ј-triiodothyronine (T 3 ) or the inactive metabolite 3,3Ј,5Ј-triiodothyronine, respectively (5). Based on both their unique functional properties and the structural information derived from cDNA sequences, three isoforms designated type I, II, and III iodothyronine deiodinases (DI, DII, and DIII, respectively) have been identified.The cDNAs for all three deiodinase isoforms contain within their coding regions an in-frame TGA triplet that has been demonstrated to code for the uncommon amino acid selenocysteine (1, 2, 4, 6, 7). This residue plays an essential role in the function of these enzymes; mutagenesis of the TGA triplet to a cysteine codon markedly reduces catalytic activity (1-3). It thus appears that the more potent nucleophilic capability of selenium, as compared with sulfur, is required for efficient deiodination. This thesis has been underscored by the identification of cDNAs coding for homologues of these enzymes from several fish, amphibian, and mammalian species (3, 8 -10). All such cDNAs isolated to date have demonstrated strict conservation of the active-site selenocysteine codon.Complementary DNAs that code for DII from Rana catesbeiana (3), rat (designated rBAT1-1), and human have been identified most recently (4). The proteins coded by these cDNAs are highly conserved; rBAT1-1 DII demonstrates 73 and 87% amino acid identity to the R. catesbeiana and human homologues, respectively. In addition, expression studies have demonstrated that these enzymes manifest all of the unique characteristics of DII (3, 4, 7).Evidence that the rBAT1-1 DII cDNA and its human and amphibian homo...
IntroductionProtein 4.2 is a major constituent of the red blood cell (RBC) membrane skeletal network, present at about 200 000 copies per RBC. 1 The protein-4.2 gene EPB42 contains 13 exons. 2,3 There are two isoforms of protein 4.2, a minor 74-kDa isoform obtained when all these exons of the gene are expressed and a 72-kDa major isoform of protein 4.2 that lacks 30 of the 33 amino acids that are encoded by exon 1. 4,5 The exact role of protein 4.2 in RBCs has not been elucidated, but protein 4.2 binds to the N-terminal cytoplasmic domain of the band-3 anion exchanger (AE1) and also interacts with ankyrin in RBCs. 6,7 The presence of band 3 is critical for the stable incorporation of protein 4.2 into the RBC membrane, since human, 8 mouse, 9 and cow 10 RBCs deficient in band 3 are also completely deficient in protein 4.2. The functional significance of the association of protein 4.2 with band 3 in RBCs is currently unclear. In liposomes containing reconstituted band 3, anion transport activity decreased in the presence of increasing amounts of protein 4.2, 11 which suggested that protein 4.2 was a negative modulator of band-3 anion exchange activity. However, band-3-mediated anion transport activity has been reported to be unaffected 12 or increased 13 in human RBCs with protein-4.2 deficiency. In contrast, the absence of protein 4.2 slightly decreased anion transport activity in protein-4.2-null mouse RBCs. 14 Protein 4.2 also associates with spectrin, 15,16 ankyrin, 7 and protein 4.1 7 in solution, although the significance of these associations in vivo has yet to be demonstrated. Recently, protein 4.2 has been shown to interact with CD47, which probably contributes to the anchoring of the Rh complex to the RBC skeleton. 17,18 Protein 4.2 can probably interact with the inner leaflet of the lipid bilayer since it is fatty acylated with myristoyl and palmitoyl chains. 19,20 The complete or nearly complete absence of protein 4.2 is associated with an atypical form of hereditary spherocytosis (HS), highlighting the important role 4.2 plays in maintaining the stability and flexibility of RBCs. To date, 9 protein-4.2 mutations have been found associated with HS. Five lead to the premature termination of translation. 17,[21][22][23][24] Other protein-4.2 variants result from missense mutations that yield amino acid substitutions: protein 4.2 Nippon (GCTϾACT; A142T 25 ; occurs sporadically in the Japanese population and has been encountered once in whites), 26 protein 4.2 Tozeur (CGAϾCAA; R310Q), 27 protein 4.2 Shiga (CGCϾTGC; R317C), 28 and protein 4.2 Komatsu (GATϾTAT; D175Y). 29 Although there is a strict correlation between the occurrence of HS and the presence of these mutations (in homozygous or compound heterozygous states), the fact that these mutations are the direct cause of the absence of protein 4.2 has not been formally established. One possibility is that these point mutations affect the binding of protein 4.2 to band 3 and therefore its function. This has proved difficult to study using biochemical We h...
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