Implication of type 3 deiodinase induction in zebrafish fin regeneration.

Thyroid hormones are critical determinants of cellular differentiation. We used the zebrafish model to evaluate the involvement of thyroid hormones in regeneration processes after caudal fin amputation. We examined early events following fin amputation, i.e., blastema formation and nerve repair by growth cone formation. Here, we show that the abolition of thyroid gland activity by methimazole treatment had no effect on blastema formation, but slowed growth cone formation of the lateral line. Conversely, the addition of exogenous thyroid hormones enhanced growth cone formation without affecting blastema formation. However, amputation triggered a strong induction in the blastema of type 3 deiodinase mRNA and enzymatic activity, which degrades thyroid hormone (TH). We therefore blocked deiodinase activity with iopanoic acid (IOP) and saw a reduction in blastema formation, suggesting that local degradation of TH is permissive for cell proliferation in the blastema. The effect of IOP on the blastema required endogenous or exogenous TH. Our findings support a model in which local degradation of TH by type 3 deiodinase is permissive for epimorphic regeneration.

Oxidative stress regulates type 3 deiodinase and type 2 deiodinase in cultured rat astrocytes.

Type 2 deiodinase (D2) and type 3 deiodinase (D3) locally achieve the determination of the concentration of T3, which binds to the thyroid hormone receptor with high affinity. D2 converts T4 into T3, and D3 degrades T4 and T3. Neurons take up T3 released by astrocytes, the main cerebral site for the D2 expression. Because oxidative stress is believed to be involved in several neurological disorders, we explored the effects of oxidative stress on D3 and D2 in primary culture of rat astrocytes. H2O2 (250 microm) increased D3 activity with maximal effects around 8 h. Stimulation of D3 activity by H2O2 was synergistic with T4, phorbol ester, and also cAMP. H2O2 (250 microm) did not affect basal D2 activity but inhibited the stimulation of D2 activity by cAMP and factors implicating cAMP-independent pathways in astrocytes, TSH, and phorbol ester. N-Acetyl cysteine and selenium repletion, which respectively increase intracellular glutathione and glutathione peroxidase, inhibited D2 and D3 regulation by H2O2, whereas L-buthionine sulfoximine, which decreases intracellular glutathione, mimicked H2O2 effects. Oxidative stress up-regulated D3 and inhibited cAMP-stimulated D2 by transcriptional mechanisms. A decrease in cAMP by oxidative stress could contribute to the inhibition of cAMP-stimulated D2. Using specific inhibitors of signaling pathways, we show that the ERK pathway was required in D2 and D3 regulation by oxidative stress and that the p38 MAPK pathway was implicated in H2O2-induced D3. We suggest that the expected decrease in T3 might modulate the cellular injury of oxidative stress in some pathological brain conditions.

Hypoxia stabilizes type 2 deiodinase activity in rat astrocytes.

T(4) activation into T(3) is catalyzed by type 2 deiodinase (D2) in the brain. The rapid induction of D2 in astrocytes by transient brain ischemia has prompted us to explore the effects of hypoxia on D2 in cultures of astrocytes. Hypoxia (2.5% O(2)) of cultured astrocytes increased D2 activity, alone or in association with agents stimulating the cAMP pathway. Hypoxia had no effect on D2 mRNA accumulation. Cycloheximide did not block the effect of hypoxia on D2 activity and D2 half-life was enhanced under hypoxia demonstrating a posttranslational action of hypoxia. Furthermore, the D2 activity increase by hypoxia was not additive with the increase promoted by the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). This strongly suggests that hypoxia leads to stabilization of D2 by slowing its degradation by the proteasome pathway. Hypoxia, in contrast to MG132, did not block the T(4)-induced D2 inactivation. A contribution of prolyl hydroxylase to the hypoxia effects on D2 was also suggested on the basis of increased D2 activity after addition of different prolyl hydroxylase inhibitors (cobalt chloride, desferrioxamine, dimethyloxalylglycine, dimethylsuccinate). Specific inhibitors of ERK, p38 MAPK, or phosphatidylinositol 3-kinase pathways were without any effect on hypoxia-increased D2 activity, eliminating their role in the effects of hypoxia. Interestingly, diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase inhibited the hypoxia-increased D2 indicating a role for some reactive oxygen species in the mechanism of D2 increase. Further studies are required to clarify the precise molecular mechanisms involved in the D2 stabilization by hypoxia.

Induction of type 2 iodothyronine deiodinase in astrocytes after transient focal cerebral ischemia in the rat.

This study investigated the expression of deiodinases of thyroid hormones in the rat brain after transient occlusion of the middle cerebral artery. The activity of type 2 deiodinase (D2), which catalyzes the deiodination of thyroxine into the more active thyroid hormone 3,5,3'-triiodothyronine, was strongly increased by cerebral ischemia at 6 and 24 hours in the striatum and at 24 hours in the cerebral cortex. The activity of type 3 deiodinase, which catalyzes the inactivation of thyroid hormones, was not affected by ischemia. In situ hybridization showed, as soon as 6 hours, an upregulation of the expression of D2 mRNA in the ipsilateral striatum, which disappeared at 24 hours. In the ipsilateral cortex, the induction of D2 mRNA started at 6 hours, was increased at 24 hours and finally declined at 72 hours. These results were confirmed by reverse transcription-PCR for D2 mRNA in the striatum and cerebral cortex. The upregulation of D2 mRNA after ischemia was mainly localized in astrocytic cell bodies. These results show that D2 is rapidly induced in astrocytes after ischemic stroke. Future work will include the exploration of the role of the upregulation of this enzyme, responsible for local 3,5,3'-triiodothyronine production as a neuroprotective mechanism in the brain.

Role of redox status on the activation of mitogen-activated protein kinase cascades by NSAIDs.

High concentrations of non steroidal antiinflammatory drugs (NSAIDs) exert preventive effects against carcinogenesis. Their molecular mechanism of action remains to be elucidated. Based on previous reports with salicylate, we have made the hypothesis that various NSAIDs can activate the mitogen-activated protein kinases (MAPK). Moreover, we tested the idea that NSAIDs act by increasing the effects of oxidative stress. We report that in human colorectal carcinoma cells NSAIDs stimulated the three families of MAPK, extracellular regulated kinases, c-Jun N-terminal kinases, p38 MAPK and that this stimulation is prevented by N-acetyl cysteine. In cultured astrocytes, a biological system less sensitive to oxidative stress, we show that a short treatment by NSAIDs strongly activated the three MAP kinases in the presence of H(2)O(2). A 25 microM H(2)O(2), unable to stimulate by itself the MAP kinases, promote an almost complete activation of MAP kinases in the presence of NSAIDs. The activation of MAP kinases by H(2)O(2) and NSAIDs was suppressed by quinone reductase inhibitors, suggesting that "redox cycling" was involved in the activation mechanisms of MAP kinases by H(2)O(2) and NSAIDs. The mobility on SDS-PAGE of the apoptosis signal-regulating kinase, which activates C-Jun N-terminal kinases and p38 MAPK cascades, was reduced by H(2)O(2) and NSAIDs, suggesting, that H(2)O(2) and NSAIDs activated apoptosis signal-regulating kinase by increasing its state of phosphorylation. In conclusion, we demonstrate that various NSAIDs can activate the three families of MAP kinases and that this activation depends on the presence of reactive oxygenated species. These results give a new insight into the mechanism of the action of NSAIDs.

MAP kinase activation by fluoxetine and its relation to gene expression in cultured rat astrocytes.

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.

Bacterial lipopolysaccharide induces type 2 deiodinase in cultured rat astrocytes.

In the brain, 3,5,3'-triiodothyronine, which binds to the thyroid hormone receptor with high affinity, is locally generated from thyroxine by type 2 iodothyronine deiodinase (D2) expressed mainly in astrocytes and tanycytes. We have investigated the effects of bacterial lipopolysaccharide (LPS) on D2 in cultured rat astrocytes. LPS induced D2 activity with a lag-time of 4-8 h and a maximum at 24 h. LPS also promoted D2 mRNA accumulation. Glucocorticoids enhanced both the basal and LPS-stimulated D2 activity and mRNA accumulation. These glucocorticoid effects were blocked by the glucocorticoid receptor antagonist RU486. Our results obtained with different specific signaling pathway inhibitors indicated that D2 induction by LPS required ERK and p38-MAPK signaling pathways. NF-κB inhibitor sulfasalazine blocked the effects of LPS on both D2 activity and mRNA accumulation. Hence, D2 induction by LPS appeared to implicate NF-κB pathway in astrocytes. NF-κB responsiveness of the rat dio2 gene was studied in astrocytes with dio2 5'-flanking region promoter assays. The long form of the dio2 promoter was transactivated by NF-κB. CCAAT/enhancer-binding protein ß, which is upregulated by LPS in astrocytes, increased the transcriptional activity of the dio2 promoter in its long or truncated forms containing CCAATs. Our observations, which demonstrate D2 induction by LPS in astrocytes and specify some characteristics of D2 induction mechanism, support the possible implication of brain D2 in adaptative responses to an infectious stress.

Protein-4.2 association with band 3 (AE1, SLCA4) in Xenopus oocytes: effects of three natural protein-4.2 mutations associated with hemolytic anemia.

We have investigated the effects of coexpression of protein 4.2 and three protein-4.2 variants with band 3 in the Xenopus oocyte expression system. Normal protein 4.2 increased band-3-specific chloride transport in the oocytes. Protein 4.2 also coimmunoprecipitated with band 3 and colocalized with band 3 at the oocyte plasma membrane. The increase in band-3-mediated chloride transport and coimmunoprecipitation of protein 4.2 required the presence of the N-terminal cytoplasmic domain of band 3. Protein 4.2 also localized to the oocyte plasma membrane in the absence of band 3. The protein-4.2 variants 4.2 Tozeur (R310Q) and 4.2 Komatsu (D175Y) had impaired ability to bind to band 3 and these variants did not localize to the oocyte plasma membrane when expressed on their own or when coexpressed with band 3. Unexpectedly, 4.2 Nippon (A142T) behaved similarly to normal protein 4.2. In the absence of a crystal structure of protein 4.2, we propose a homology model of protein 4.2 based on the structure of the sequence-related protein transglutaminase. Using our results in oocytes and this homology model we speculate how these mutations affect protein 4.2 and result in hereditary spherocytosis.

MAP kinase cascades are activated in astrocytes and preadipocytes by 15-deoxy-Delta(12-14)-prostaglandin J(2) and the thiazolidinedione ciglitazone through peroxisome proliferator activator receptor gamma-independent mechanisms involving reactive oxygenated species.

15-Deoxy-Delta(12-14)-prostaglandin J(2) (dPGJ2) and thiazolidinediones are known as ligands for the peroxisome proliferator activator receptor gamma (PPAR gamma) 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 microm). 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 gamma. Ciglitazone (20 microm), 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 gamma, rosiglitazone, does not activate MAP kinases, indicating that the effect of ciglitazone on MAP kinases is independent of PPAR gamma. 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 gamma-independent mechanisms involving ROS.