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Endocrinology, doi:10.1210/en.2007-0625
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Endocrinology Vol. 148, No. 10 4745-4753
Copyright © 2007 by The Endocrine Society

Hypoxia Stabilizes Type 2 Deiodinase Activity in Rat Astrocytes

Audrey Lamirand, Gilles Mercier, Martine Ramaugé, Michel Pierre and Françoise Courtin

Unité Mixte de Recherche 854 Institut National de la Santé et de la Recherche Médicale and Université Paris-Sud 11, 94276 Le Kremlin-Bicêtre, France

Address all correspondence and requests for reprints to: Françoise Courtin, Unité Mixte de Recherche 854 Institut National de la Santé et de la Recherche Médicale, 80 Rue du Général Leclerc and Paris-Sud 11, 94276 Le Kremlin-Bicêtre, France. E-mail: courtin{at}kb.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T4 activation into T3 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% O2) 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 T4-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONES (TH) are essential for development and maturation of the central nervous system (1, 2, 3, 4). In the newborn, prolonged hypothyroidism leads to irreversible effects on the brain such as mental retardation or cretinism, resulting from defects of cell migration, differentiation, myelination, and synaptogenesis. In adults, TH impairments can lead to neurological and psychiatric disorders that can be treated by TH replacement (5). Some studies suggest a neuroprotective role of TH after ischemia in dog or rat (6, 7). TH appear to be crucial regulators of neural stem cell proliferation and oligodendrocyte precursor cell fate not only during development but also in the mature brain and after injury (8, 9, 10). TH administration enhances remyelination in chronic demyelinating inflammatory disease (8).

The main product of the thyroid gland is the prohormone T4, which can be activated by T3, the hormone form that binds the thyroid hormone receptor with high affinity. In the brain, most T3 is synthesized locally by deiodination of T4 to T3 catalyzed by the type 2 iodothyronine deiodinase (D2) (11). Brain D2, mostly expressed in astrocytes, is a selenoprotein like the other deiodinases (12, 13, 14, 15, 16, 17). Cerebral T3 concentration is also regulated by the type 3 deiodinase (D3), which catalyzes the deiodination of TH into inactive metabolites (16, 17). Thyroid status regulates the expression of brain deiodinases to maintain brain T3 concentration (16, 17). The temporal and spatial pattern of D2 and D3 expression in the central nervous system appears to be highly regulated, especially during development (13, 14, 15, 18, 19). Important accumulation of D2 mRNA in astrocytes has been described after brain injury (20, 21). A rapid increase in D2 activity and mRNA accumulation after ischemic stroke raises the question of the neuroprotective role of D2 (21). No regulation of D3 expression was observed after ischemic stroke (21). The mechanism of D2 induction after transient focal cerebral ischemia remains unknown. D2 multiregulation has been well established in cultured rat astrocytes. cAMP, TSH, glucocorticoids, phorbol esters, fibroblast growth factors, and selenium increase D2 activity (15, 22, 23, 24, 25, 26). cAMP and selenium increase D2 activity, respectively, by transcriptional and translational mechanisms in astrocytes (15) as in other cell types (16). T4 and rT3 inhibit D2 by posttranslational mechanisms in astrocytes (27) and other cell types (16). Because ischemia promotes oxygen deprivation, we investigated in the present study the effects of hypoxia on D2 and D3 expression in cultured astrocytes. We demonstrate that hypoxia leads to stabilization of D2 by slowing its degradation by the proteasome pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and animals
T4, T3, dithiothreitol (DTT), forskolin, cycloheximide, actinomycin D, dimethyl succinate (DMS), cobalt chloride (CoCl2), desferrioxamine (DFO), N-acetyl cysteine (NAC), SB203580, diphenyleneiodonium (DPI), and antibiotics were obtained from Sigma Aldrich Co. (St. Louis, MO). Dimethyloxalylglycine (DMOG) was obtained from Cayman Chemical (Ann Arbor, MI). Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) was obtained from Calbiochem (San Diego, CA). U0126 and wortmannin were purchased from Biomol (Plymouth Meeting, PA). 12-0-Tetradecanoylphorbol-13-acetate (TPA) was obtained from Alexis (San Diego, CA). Sodium selenite was purchased from Merck (Darmstadt, Germany). [125I]T3 (3 mCi/µg) and [125I]T4 (1.5 mCi/µg) were purchased from Amersham International (Buckinghamshire, UK). Sprague Dawley rats were purchased from Iffa-Credo (L’Albresle, France). Fetal calf serum (FCS) and culture media were obtained from Life Technologies (Grand Island, NY), and culture dishes were obtained from Nunclon (Roskilde, Denmark).

Cell culture conditions
Brains were removed from 2-d-old Sprague Dawley rats and cleaned of meninges and blood vessels. The two cerebral hemispheres were then dissociated to form a cell suspension by passage through a nylon mesh (82 µm pore size) into 40 ml DMEM supplemented to 6 g/liter glucose, 2.4 g/liter sodium bicarbonate, antibiotics (100 U/ml penicillin, 100 µg/liter streptomycin, and 0.25 µg/liter amphotericin B), and 10% FCS (DMEM/FCS). Aliquots (3 ml) of this cell suspension were placed in 60-mm petri dishes and the cultures were maintained at 37 C under more than 90% humidity, 5% CO2, and 95% air, i.e. 21% O2. The medium was changed every 2–3 d until cells reached confluency at approximately 10 d. At this stage the DMEM/FCS was removed, and the cells were washed with a chemically defined medium that consisted in a 1:1 mixture of DMEM and Ham’s F-12 medium (DMEM/F12) supplemented to 4.5 g/liter glucose, 1.8 g/liter sodium bicarbonate, and the antibiotics listed above. The cells were then cultured for 3 additional days in DMEM/F12 supplemented with 30 nM sodium selenite, 10 µg/ml insulin, and 10 µg/ml transferrin followed by 1 additional day in DMEM/F12 supplemented with 30 nM sodium selenite and 10 µg/ml transferrin. Astrocytes were treated with the test agents for the times and at the concentrations indicated in each experiment.

Hypoxia treatment
Hypoxia was achieved inside an incubator chamber with an O2 sensor-controlling infusion of N2 (ProOx instrument; BioSpherix, Redfield, NY). O2 concentrations (1 and 2.5%) were those generally used for hypoxia studies in cell cultures. Other parameters were those in normoxia (90% humidity, 5% CO2, 37 C).

D2 mRNA quantitation by RT-PCR assays
Total RNA was extracted using a GenElute mammalian total RNA mimiprep kit (Sigma Aldrich) and quantified by absorption at 260 nm. Subsequently, cDNA was synthesized by reverse transcription using the Muloney murine leukemia virus reverse transcriptase system (Invitrogen Corp., Carlsbad, CA). The cDNAs were amplified in a real-time PCR using the Taqman gene expression assays with the designing primers for D2 and 18rRNA as an endogenous control (Applied Biosystems, Foster City, CA). The generated D2 and 18S cDNA levels were measured using the ABI Prism 7000 sequence detection system and the relative standard curve method. Briefly, all genes were quantified from a standard curve representing five-point serial dilutions of mixed experimental and control cDNA, which were analyzed and used as calibrators for the quantification of the product generated in the exponential phase of the amplification curve. Both standards and samples were run in duplicate. Typically the equivalent cDNA of 20 ng RNA was used for the real-time PCR of each sample. The R2 was greater than 0.99 for all standard curves.

D2 and D3 assays
At the time of harvesting, the medium was aspirated, and the cells were rinsed with 3 ml ice-cold PBS on ice. Culture dishes containing the cells were then frozen at –80 C for later processing and analysis. This involved placing the plates on ice, scraping the content of each dish into 0.4 ml sample buffer [20 mM HEPES, 2 mM DTT, and 0.25 M sucrose (pH 7.4)], and disrupting the cells by sonication. D2 activity was measured by incubating aliquots of the cell sonicate in an 80 µl final volume of 20 mM HEPES (pH 7.4), containing 20 mM DTT, 50 nM T3, and 1 nM [125I]T4 for 20–60 min at 37 C. Reactions were stopped by adding 10 µl of 10 M NH4OH containing 10 µM T3 and 10 µM T4. The [125I]T3 produced was separated from [125I]T4 by descending paper chromatography (12). Then the radioactive products were counted for determination of D2 activity, expressed as femtomoles of T3 per minute per milligram of protein. Kinetic analysis was performed in sonicates of forskolin-treated cells using 0.05–2 nM [125I]T4 as substrate and 20 mM DTT as cofactor. Usual variations in basal and forskolin-stimulated D2 activities were observed from one experiment to another. For D3 activity assays, homogenates were incubated at 37 C for 60 min in a final volume of 80 µl containing 20 mM HEPES buffer (pH 7.4), 20 mM DTT, and 5 nM [125I]T3. Reactions were stopped by adding 10 µl of 10 M NH4OH containing 10 µM T3 and 10 µM T4. The [125I]3,3'-diiodothyronine produced was separated from [125I]T3 by descending paper chromatography (12). Then the radioactive products were counted for determination of D3 activity, expressed as femtomoles of [125I]3,3'-diiodothyronine per minute per milligram of protein. For D2 and D3 assays, deiodination was linear with respect to both protein concentration and incubation time and the quantity of protein assayed was adjusted to ensure that less than 30% of the substrate was consumed. We controlled that T4 present in the extracts does not interfere with the assay.

Protein determination
The protein content of cell sonicates was determined by the method of Bradford (28) using BSA as standard.

Statistical analysis
All data were analyzed by a commercially available program (GraphPad Prism 3.0; GraphPad Inc., San Diego, CA). Data were processed using one- or two-way ANOVA, followed by Bonferroni post hoc test in the figures. Student’s t test was also used when indicated in the text. Statistical significance was noted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of hypoxia on the activity and mRNA accumulation of D2
Effects of hypoxia on the D2 activity and mRNA accumulation were investigated in primary cultures of astrocytes by maintaining cells in normoxia (21% O2) or hypoxia (2.5% O2) for 10 h (Fig. 1Go, A and B). Astrocytes were cultured in basal conditions or treated by agents elevating intracellular cAMP, which is known to induce D2 in rat astroglial cells (22, 23). In cells cultured in normoxia, a strong increases in D2 activity and D2 mRNA accumulation were observed 12 h after addition of 10 µM forskolin, which directly and durably activates adenylate cyclase generating cAMP. Figure 1AGo shows that hypoxia increased the D2 activity by around 1.5 in basal conditions, but this difference was significant only by t' test analysis (P < 0.05). The range of D2 activity in basal conditions were very low and measurement relatively imprecise. Hypoxia increased the D2 activity by around 3-fold in cAMP-stimulated conditions. Similarly, hypoxia for 10 h increased the D2 activity 3-fold in astrocytes that were pretreated for 2 h by 10 µM isoproterenol, a ß-adrenergic agonist transiently elevating cAMP (not shown). This strongly suggests that durable increase in cAMP appears not fundamental in the hypoxia effect. In contrast, hypoxia did not modify D2 mRNA accumulation in basal or stimulated conditions with forskolin (Fig. 1BGo) or isoproterenol (not shown). In addition, the hypoxia-stimulated increase in D2 activity was time dependent. The hypoxia-stimulated increase in D2 activity was visible as soon as 2 h (see Figs. 1CGo and 3BGo). In Fig. 1CGo, the difference at 2 h was significant only by t test analysis (P < 0.05). The hypoxia-increased D2 activity was variable from one experiment to another (1.5- to 7-fold after 8 h hypoxia with 2.5% O2, not shown). The increase in D2 activity by hypoxia was reversed after 2 h in normoxia (not shown). The Michaelis constant for T4 remained unchanged (around 1 nM) under hypoxic conditions (Fig. 1DGo) for cAMP-induced D2 activity. Therefore, hypoxia did not modify the D2 affinity for its substrate. For the following experiments, D2 activities were always stimulated by forskolin to do precise measurement of D2 activity as previously done in astrocytes (27), and the presented experiments reflected three independent experiments.


Figure 1
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  		FIG. 1. Hypoxia increases D2 activity without affecting D2 mRNA accumulation. A and B, Astrocytes were induced or not with 10 µM forskolin (Fk) for 12 h before harvesting and were kept under normoxia or under hypoxia (2.5% O2) for the last 10 h. D2 activity (A) and D2 mRNA accumulation (B) were measured as described in Materials and Methods. C, Kinetics of D2 activity induction by hypoxia. Astrocytes were treated with 10 µM forskolin for 8 h before harvesting and kept under 2.5% O2 hypoxia for the times indicated before harvesting. D2 activity was measured as described in Materials and Methods. D, Lineweaver-Burk plots of D2 activity in cellular homogenates of astrocytes treated with 10 µM forskolin for 12 h before harvesting and kept under hypoxia (2.5% O2) ({circ}) or normoxia (bullet) during the last 10 h. D2 activity was measured in triplicate for different concentrations of T4. Data are the means ± SD of results obtained from three dishes in A–C. *, P < 0.05 and ***, P < 0.001 vs. the respective controls in normoxia.

 

Figure 3
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  		FIG. 3. Hypoxia increases D2 activity by a posttranslational mechanism. A, Four hours after addition of 10 µM forskolin, astrocytes were incubated under hypoxia (2.5% O2) ({circ}) or normoxia (bullet). Four hours after the beginning of hypoxia, 18 µM cycloheximide was added, for the times indicated, in cells maintained under normoxia or hypoxia. D2 activity is expressed as the mean percentage of the remaining enzyme activity. B, Astrocytes were treated for 10 h with 10 µM forskolin before harvesting and kept under hypoxia (2.5% O2) for the last 2 h when indicated. When indicated, 18 µM cycloheximide (CHX) and 5 µM MG132 were added, respectively, 20 and 10 min before hypoxia. C, Astrocytes were treated with 10 µM forskolin for 8 h before harvesting and, when indicated, kept under hypoxia (1% O2) for the last 4 h or treated with 5 µM MG132 for the last 2 h 20 min. T4 (1 nM) was added, when indicated, in the medium for the last 2 h before harvesting. D2 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. the respective controls in normoxia.

 
Effects of hypoxia on the activity and mRNA accumulation of D3
We also followed the effects of hypoxia (2.5% O2) for 10 h on D3 activity in basal or stimulated conditions. In cells cultured in normoxia, D3 activity was not affected by 10 µM forskolin considered in any case as a poor inducer of D3 activity (29) but was induced by 0.1 µM TPA (Fig. 2AGo) or 10 nM T3 (Fig. 2BGo) as previously described (29, 30). We found that hypoxia did not modify D3 activity and D3 mRNA (not shown) in cultured astrocytes in basal conditions or in presence of forskolin, TPA, or T3.


Figure 2
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  		FIG. 2. Hypoxia does not affect D3 activity. Astrocytes were treated or not before harvesting with 10 µM forskolin (Fk) for 12 h, 0.1 µM TPA for 12 h (A), or 10 nM T3 for 24 h (B) and kept under normoxia or 2.5% O2 hypoxia for the last 10 h when indicated. D3 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes.

 
Posttranslational effect of hypoxia on D2
The hypoxia-increased D2 activity without changes in D2 mRNA accumulation led us to explore whether hypoxia had an effect on the half-life of D2 activity. We compared D2’s half-life in normoxia and hypoxia by blocking protein synthesis with cycloheximide. In astrocytes stimulated by forskolin, the D2 half-life was 25–30 min in normoxia and increased to 85–90 min in hypoxia (Fig. 3AGo). The short half-life of D2 has been described in rat astrocytes (27) in GH3 cells (31) as well as rat pituitary tumor cells (32). In the latter cells, the short half-life of D2 activity has been shown to result from D2 degradation by the ubiquitin-proteasome pathway (32). We also showed that in astrocytes, blocking the proteasome degradation pathway with 5 µM MG132, an inhibitor of the protease activity of the proteasome, increased the D2 activity in the absence or presence of cycloheximide (Fig. 3BGo). The D2 activity increase caused by 2.5% O2 hypoxia was also observed in the presence of cycloheximide, indicating a posttranslational mechanism for hypoxia as for MG132. Moreover, the increase in D2 activity by hypoxia was not additive with the D2 activity increase by MG132, suggesting that hypoxia leads also to stabilization of D2 by slowing its degradation by the proteasome pathway. In addition, we explored the effects of hypoxia on the well-known down-regulation of D2 activity by its substrate, also described in rat astrocytes (27). Substrate accelerates the degradation of D2 through the ubiquitin-proteasome pathway in rat pituitary tumor cells (32). A 1% O2 hypoxia had no effect on the inhibition of D2 by T4, whereas the proteasome inhibitor MG132 effectively blocked the D2 inhibition by T4 (Fig. 3CGo). Therefore, hypoxia seems to act on basal but not T4-accelerated D2 degradation.

Effect of glucose depletion
Because the accumulation of hypoxia inducible factor (HIF), a transcriptional factor responsible of numerous cell responses under hypoxia (33), has been described to require glucose availability (34), astrocytes were cultured in a DMEM medium depleted or not of glucose for 24 h. A decrease in D2 activity was observed in glucose-depleted conditions (Fig. 4Go), which was significant by t' test analysis (P < 0.05). However, astrocyte D2 activity remained able to respond to hypoxia with apparently the same increase (the ratios between the activities in the presence and absence of hypoxia were not significantly different by t' test analysis (217 ± 9 in glucose free conditions and 243 ± 23 glucose supplemented conditions).


Figure 4
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  		FIG. 4. Glucose depletion does not prevent the effect of hypoxia on D2 activity. Astrocytes were cultured in the absence or not of 25 mM glucose for the last 24 h. Astrocytes were treated for 9 h with 10 µM forskolin before harvesting and incubated under hypoxia (2.5% O2) for the last 8 h when indicated. D2 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes. ***, P < 0.001 vs. the respective controls in normoxia.

 
Effects of chemical hypoxia
The increase in D2 activity by reducing oxygenation prompted us to test whether agents promoting chemical hypoxia were also able to increase D2 activity. The iron chelator DFO, CoCl2, DMOG, or DMS mainly mimics O2 depletion on the accumulation of HIF-1{alpha} (35, 36, 37, 38, 39). Their main mechanism of action is the inhibition of Fe(II)-2-oxoglutarate-dependent dioxygenases, including proline hydroxylase and factor inhibiting HIF (40). Figure 5Go, A and B, shows that D2 activity transiently increased after DFO or CoCl2 treatment for 2–4 h and returned to initial levels after 8 h. DMOG, which is a cell-permeable precursor of an inhibitor of prolyl-hydroxylase (35), induced D2 activity at 0.1 and 0.2 mM for 2 h (Fig. 5CGo). As for DFO and CoCl2, DMOG-increased D2 activity disappeared after 8 h (not shown). At higher concentration, 0.5 mM, DMOG did not increase D2 activity. DMS, which penetrates the cell membrane and is metabolized to succinate (41), promoted a progressive increase in D2 activity from 8 h until 24 h (Fig. 5DGo).


Figure 5
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  		FIG. 5. Effect of prolyl hydroxylase inhibitors on D2 activity. Astrocytes were treated with 10 µM forskolin for 8 h before harvesting in A–D. Then 100 µM DFO (A) and 100 µM CoCl2 (B) were added for the times indicated before harvesting. DMOG (C) was added at different concentrations for the last 2 h (C) in 0.1% DMSO; 20 mM DMS (D) was added for the times indicated before harvesting. D2 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. the respective controls without inhibitor treatment.

 
Protein kinase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase implication in hypoxia-increased D2 activity
Because the activation of several kinases has been demonstrated under hypoxic conditions (42), effects of hypoxia on D2 activity were studied in the presence of wortmannin, SB203580, and U0126, inhibitors of p38 MAPK, phosphatidylinositol 3-kinase (PI3 kinase), and ERK pathways (Fig. 6Go). The hypoxia significantly increased D2 activity in all pretreatment situations (two-way ANOVA analysis; P < 0.001). Bonferroni posttest was significant (Fig. 6Go) for dimethylsulfoxide (DMSO) (vehicle), wortmannin, and SB203580 but not for U0126 for which difference was, however, significant by t' test analysis (P < 0.05). The increase in D2 activity by hypoxia was similar in the presence of DMSO, wortmannin, SB203580, or U0126. Indeed, the ratios between the activities in the presence and absence of hypoxia were not significant by one-way ANOVA analysis (272 ± 70 in presence of DMSO, 254 ± 101 in presence of wortmannin, 235 ± 52 in presence of SB203580, and 198 ± 54 in presence of U0126). On the other hand, some studies have implicated the generation of reactive oxygen species by NADPH oxidase as mediators of HIF-1{alpha} protein stabilization and activation (43) and HIF-2{alpha} expression (44). To analyze the effect of reactive oxygen species generation by NADPH oxidase, we used DPI, an inhibitor of flavoprotein containing oxidases. DPI at a concentration that did not affect mitochondrial oxidase in astrocytes (45) inhibited the D2 activity increase by hypoxia (Fig. 7AGo), suggesting a role for O2.– in the hypoxic effect on D2 stabilization. NAC (2–20 mM) was unable to reduce the hypoxic effect on D2 activity, eliminating a role for H2O2 (Fig. 7BGo).


Figure 6
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  		FIG. 6. Effect of hypoxia does not involve PI3 kinase, p38 MAPK, or ERK pathways. Astrocytes were treated with 10 µM forskolin for 8 h before harvesting and kept under hypoxia (2.5% O2) for the last 4 h when indicated. Then 0.1% DMSO (vehicle), 100 nM wortmannin (Wt), 5 µM SB203580 (SB), or 5 µM U0126 were added 20 min before normoxia or hypoxia. D2 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes. *, P < 0.05 and **, P < 0.01 vs. the respective controls in normoxia.

 

Figure 7
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  		FIG. 7. Involvement of NADPH oxidase in the D2 increase caused by hypoxia. Astrocytes were treated with 10 µM forskolin for 8 h before harvesting and kept under hypoxia (2.5% O2) for the last 4 h when indicated. Then 30 min before normoxia or hypoxia, DMSO (0.1%) or vehicle of DPI or DPI (0.5 µM) was added in A, whereas indicated concentrations of NAC were added in B. D2 activity was measured as described in Materials and Methods. Data are the means ± SD of results obtained from three dishes. ***, P < 0.001 vs. the respective controls in normoxia.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report on D2 regulation by low oxygen tension or hypoxia in any cell culture model. So in primary cultures of astrocytes, hypoxia (1–2.5% O2), compared with normoxia (21% O2), increases D2 activity without affecting D2 mRNA accumulation. The enhancement of D2 half-life under hypoxia demonstrates a posttranslational action of hypoxia. D2 is a short half-life protein degraded through the ubiquitin-proteasome pathway (46). Interestingly, we observed that the increase in D2 activity by hypoxia was not additive with that promoted by the proteasome inhibitor MG132, strongly suggesting that hypoxia stabilizes D2 by slowing its degradation by the proteasome pathway.

The absence of a hypoxic effect on D2 mRNA allows elimination of a direct role of HIF, a transcriptional factor that plays a crucial role in mediating cellular responses to oxygen in various cell types (33, 40) including astrocytes (47). HIF-1 is an ubiquitous heterodimeric protein subjected to rapid turnover under normoxic conditions, whereas hypoxia inhibits its degradation leading to the accumulation of the subunit HIF-1{alpha}. So hypoxia stabilizes D2 like HIF-1{alpha} by slowing their degradation through the ubiquitin-proteasome pathway, although their respective turnovers in normoxia are different: less than 5 min for HIF-1{alpha}, the key oxygen sensor, and 20–60 min for D2 in various cell types (46) including in astrocytes (15).

Previously an increase in D2 half-life has been described in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in astrocytes as well as rat pituitary tumor cells (27, 32). CCCP is a protonophore promoting ATP depletion that effectively decreases ubiquitin-proteasome-dependent degradation pathway (48). This CCCP-induced increase in D2 half-life was associated with a modest reduction of D2 activity (27, 32), which might be secondary to the decrease in protein synthesis promoted by ATP depletion (49). So it seems unlikely that ATP depletion contributes notably to both hypoxia-increased D2 activity and half-life. Indeed, ATP depletion is not important after hypoxia in astrocytes (50). Mobilization of glycogen stores limits the glucose depletion in astrocytes (50, 51). Glucose depletion for 4 h has been reported to have no effect on D2 half-life in astrocytes (27). Moreover, we observed that glucose depletion for 24 h reduces D2 activity in normoxia but maintains the D2 hypoxia response in astrocytes. Similarly, 22B human cancer cells cultured in a glucose-free medium are still able to markedly induce HIF-1 under hypoxia (52).

D2 is an endoplasmic reticulum resident protein (53), as suggested by an early study using cell fractionation of anterior pituitary and D2 activity measurements (54). D2 degradation is catalyzed by an estrogen receptor-associated degradation process (32, 55), i.e. D2 is selectively targeted for ubiquitination and subsequent proteasomal degradation (32, 55, 56). According to the classical model, ubiquitination occurs via a multistep process involving the actions of enzymes including ubiquitin-activating enzyme, ubiquitin conjugases, and ubiquitin ligases (E3). The basal and substrate-accelerated human D2 degradation require ubiquitin-activating enzyme (56), UBC7 or UBC6, which are estrogen receptor-associated degradation-associated ubiquitin conjugases (57, 58). Ubiquitination of D2 could occur at a lysine residue (15 residues in D2 at the C terminal) as for most proteins (59). D2 is ubiquitinated by a catalytic core complex, with WD repeat and SOCS box-containing-1 (WSB-1) functioning as an E3 ubiquitin ligase subunit and interacting with an 18-amino acid instability loop in D2 (60). The removal of the N-terminal six amino acids of the D2 loop prolongs D2’s half-life by impairing its interaction with WSB-1 but does not eliminate the susceptibility to T4 exposure or prevent proteasomal degradation, indicating the existence of different addressing mechanisms to the proteasome (61). This conclusion is strengthened by our observation of the action of hypoxia on basal D2 degradation but not T4-accelerated degradation in astrocytes. We controlled, by using the proteasome inhibitor MG132, that in astrocytes as in all tested cell models, both D2 degradation pathways are mediated by the proteasome.

Once ubiquitinated, D2 becomes inactive and then may potentially be deubiquitinated (with reactivation of the enzyme) or degraded by the proteasome (46). The balance between ubiquitination and deubiquitination is determined, at least in part, by the activity of von Hippel-Lindau protein-interacting deubiquitinating enzymes (VDU1 and VDU2, respectively), which interact with D2 (62). VDU1 and VDU2 themselves are degraded after ubiquitination mediated by pVHL-E3 ligase (63, 64). VDU1, but not VDU2, is markedly increased in vivo in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity (62). Oxygen might control the D2 degradation mechanism at various levels (VDU, D2 ubiquitination, etc.). A major example of oxygen control is the regulation of HIF-1{alpha} protein expression (33). HIF-1{alpha} is targeted for destruction by prolyl hydroxylation catalyzed by prolyl hydroxylases (33). HIF-1{alpha} transactivation domain is regulated by hypoxia, CoCl2, or iron chelation by DFO, suggesting a common step mechanism involved in both HIF-1{alpha} expression and activity. Indeed, the HIF-1{alpha} transactivation domain is blocked by O2-dependent hydroxylation of Asn 803 catalyzed by an asparaginyl hydroxylase (33). DMOG, DFO, or CoCl2 inhibit the Fe(II)-2oxoglutarate-dependent prolyl-4-hydroxylase domain-containing enzymes including HIF prolyl 4-hydroxylase and collagen prolyl 4-hydroxylase and also asparaginyl hydroxylase (65). The increase in D2 activity by hypoxia, DFO, CoCl2, or DMOG also suggest the implication of protein hydroxylation in stabilization of D2 activity by hypoxia. DMS, a membrane-permeable precursor of succinate, which also promotes HIF-1{alpha} accumulation by prolyl 4-hydroxylase inhibition (38), also increases D2 activity, suggesting a link between metabolism and D2. No oxygen-dependent degradation domain (LXXLAP) recognized by HIF prolyl hydroxylase has been identified in D2 or VDU, suggesting that other members of the Fe(II)-2oxoglutarate-dependent prolyl-4-hydroxylases family could be involved as demonstrated for inhibitory-{kappa}B (66).

Hypoxia-dependent increase in D2 activity was not affected by inhibitors of PI3 kinase, ERK, and p38 MAPK pathways, eliminating the implication of these kinases that have been reported to modulate hypoxia-dependent HIF accumulation and activation (42, 67, 68). The implication of NADPH oxidase has also been reported in HIF-1{alpha} accumulation and gene induction by hypoxia (43, 69) and more recently in HIF-2{alpha} expression (44). A role of NADPH oxidase in the hypoxia-dependent increase in D2 activity is also strongly suggested by our present observations. Indeed, the inhibition of NADPH oxidase by DPI abolishes the increase in D2 activity by hypoxia. At 0.5 µM, DPI inhibits NADPH oxidase in cultured astrocytes without affecting mitochondrial dehydrogenase (45). Surprisingly, the antioxidant NAC, which reduces H2O2 by increasing the most efficient endogenous antioxidant glutathione (70), does not prevent the increase in D2 activity by hypoxia. NAC is probably unable to modify the concentration of O2.– generated by NADPH oxidase in astrocytes as described in macrophages (71, 72). NAC also does not prevent hypoxia-dependent HIF-1{alpha} stabilization (73). NADPH is conserved in astrocytes exposed to oxygen deprivation or oxygen and glucose deprivation (74). This is probably correlated to the glycogen store mobilization in astrocytes (75). The implication of O2.– in the hypoxia-dependent D2 stabilization will be confirmed by further investigations (half-life studies, O2.– scavenging by the use of super oxide dismutase, etc.). Further works are required to elucidate the mechanisms of D2 stabilization by hypoxia including the role of NADPH oxidase and prolyl hydroxylases.

The present study contributes to the understanding of the regulation of D2 activity after transient focal ischemia in the rat brain (21). D2 mRNA accumulation is not induced in our cell culture model by hypoxia, although it is rapidly observed after ischemia. So D2 stabilization by hypoxia may potentiate D2 activity induction during ischemia and maintain D2 activity after the D2 mRNA fall, as observed 24 h after ischemia in striatum (21). Moreover, hypoxia does not block D2 down-regulation by T4, a major control of T3 production in vivo. No D3 activity induction was associated with the D2 increase by ischemia in vivo (21). Hypoxia was also unable to modify D3 activity, alone or in association with different inducers of D3 as phorbol ester or T3 in cultured astrocytes. Thus, hypoxia might increase T3 production without affecting T3 degradation in astrocytes. After release from astrocytes, T3 is taken up by neurons by a process most likely mediated by monocarboxylate transporter-8 MCT8 (76). Increase in T3 production by hypoxia might constitute a neuroprotective effect during ischemia. For example, T4 administration, in contrast to T3 administration, provides protection against global ischemia in dog (7). In the future, it will be interesting to generalize our observations to other culture systems and other pathological situations of the brain implicating hypoxia as, for example, strokes and tumor development.


   Acknowledgments
 
We thank A. Dessouroux, P. Lière, and K. Rajkowski (Unité Mixte de Recherche 788 Institut National de la Santé et de la Recherche Médicale and Paris-Sud 11, Le Kremlin-Bicêtre, France); C. Dupuy (Centre National de la Recherche Scientifique, FRE2939, Villejuif, France); and C. Ladroue and B. Gardie (Centre National de la Recherche Scientifique, FRE2939, Laboratoire Ecole Pratique des Hautes Etudes de Génétique Oncologique, Villejuif, France) for their helpful advice.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online July 5, 2007

Abbreviations: CCCP, Carbonyl cyanide m-chlorophenylhydrazone; CoCl2, cobalt chloride; D2, type 2 deiodinase; D3, type 3 deiodinase; DFO, desferrioxamine; DMOG, dimethyloxalylglycine; DMS, dimethyl succinate; DMSO, dimethylsulfoxide; DPI, diphenyleneiodonium; DTT, dithiothreitol; E3, ubiquitin ligase; FCS, fetal calf serum; HIF, hypoxia inducible factor; NAC, N-acetyl cysteine; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; NADPH, nicotinamide adenine dinucleotide phosphate oxidase; PI3 kinase, phosphatidylinositol 3-kinase; TH, thyroid hormones; TPA, 12–0-tetradecanoylphorbol-13-acetate.

Received May 10, 2007.

Accepted for publication June 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Legrand J 1982 [Thyroid hormones and maturation of the nervous system]. J Physiol (Paris) 78:603–652[Medline]
  2. Legrand J 1986 Thyroid hormone metabolism. New York: Marcel Decker
  3. Bernal J 2002 Action of thyroid hormone in brain. J Endocrinol Invest 25:268–288[Medline]
  4. Thompson CC, Potter GB 2000 Thyroid hormone action in neural development. Cereb Cortex 10:939–945[Abstract/Free Full Text]
  5. Joffe RT, Sokolov ST 1994 Thyroid hormones, the brain, and affective disorders. Crit Rev Neurobiol 8:45–63[Medline]
  6. Rami A, Krieglstein J 1992 Thyroxine attenuates hippocampal neuronal damage caused by ischemia in the rat. Life Sci 50:645–650[CrossRef][Medline]
  7. D’Alecy LG 1997 Thyroid hormone in neural rescue. Thyroid 7:115–124[Medline]
  8. Fernandez M, Giuliani A, Pirondi S, D’Intino G, Giardino L, Aloe L, Levi-Montalcini R, Calza L 2004 Thyroid hormone administration enhances remyelination in chronic demyelinating inflammatory disease. Proc Natl Acad Sci USA 101:16363–16368[Abstract/Free Full Text]
  9. Lemkine GF, Raj A, Alfama G, Turque N, Hassani Z, Alegria-Prevot O, Samarut J, Levi G, Demeneix BA 2005 Adult neural stem cell cycling in vivo requires thyroid hormone and its {alpha} receptor. FASEB J 19:863–865[Abstract/Free Full Text]
  10. Tatsumi K, Haga S, Matsuyoshi H, Inoue M, Manabe T, Makinodan M, Wanaka A 2005 Characterization of cells with proliferative activity after a brain injury. Neurochem Int 46:381–389[CrossRef][Medline]
  11. Crantz FR, Silva JE, Larsen PR 1982 An analysis of the sources and quantity of 3,5,3'-triiodothyronine specifically bound to nuclear receptors in rat cerebral cortex and cerebellum. Endocrinology 110:367–375[Abstract/Free Full Text]
  12. Courtin F, Chantoux F, Francon J 1986 Thyroid hormone metabolism by glial cells in primary culture. Mol Cell Endocrinol 48:167–178[CrossRef][Medline]
  13. Courtin F, Chantoux F, Francon J 1988 Thyroid hormone metabolism in neuron-enriched primary cultures of fetal rat brain cells. Mol Cell Endocrinol 58:73–84[CrossRef][Medline]
  14. Guadano-Ferraz A, Obregon MJ, St Germain DL, Bernal J 1997 The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci USA 94:10391–10396[Abstract/Free Full Text]
  15. Pallud S, Lennon AM, Ramauge M, Gavaret JM, Croteau W, Pierre M, Courtin F, St Germain DL 1997 Expression of the type II iodothyronine deiodinase in cultured rat astrocytes is selenium-dependent. J Biol Chem 272:18104–18110[Abstract/Free Full Text]
  16. Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:38–89[Abstract/Free Full Text]
  17. Courtin F, Zrouri H, Lamirand A, Li WW, Mercier G, Schumacher M, Goascogne CL, Pierre M 2005 Thyroid hormone deiodinases in the central and peripheral nervous system. Thyroid 15:931–942[CrossRef][Medline]
  18. Kaplan MM, Yaskoski KA 1981 Maturational patterns of iodothyronine phenolic and tyrosyl ring deiodinase activities in rat cerebrum, cerebellum, and hypothalamus. J Clin Invest 67:1208–1214[Medline]
  19. Escamez MJ, Guadano-Ferraz A, Cuadrado A, Bernal J 1999 Type 3 iodothyronine deiodinase is selectively expressed in areas related to sexual differentiation in the newborn rat brain. Endocrinology 140:5443–5446[Abstract/Free Full Text]
  20. Zou L, Burmeister LA, Styren SD, Kochanek PM, DeKosky ST 1998 Up-regulation of type 2 iodothyronine deiodinase mRNA in reactive astrocytes following traumatic brain injury in the rat. J Neurochem 71:887–890[Medline]
  21. Margaill I, Royer J, Lerouet D, Ramauge M, Le Goascogne C, Li WW, Plotkine M, Pierre M, Courtin F 2005 Induction of type 2 iodothyronine deiodinase in astrocytes after transient focal cerebral ischemia in the rat. J Cereb Blood Flow Metab 25:468–476[CrossRef][Medline]
  22. Leonard JL 1988 Dibutyryl cAMP induction of type II 5'deiodinase activity in rat brain astrocytes in culture. Biochem Biophys Res Commun 151:1164–1172[CrossRef][Medline]
  23. Courtin F, Chantoux F, Pierre M, Francon J 1988 Induction of type II 5'-deiodinase activity by cyclic adenosine 3', 5'-monophosphate in cultured rat astroglial cells. Endocrinology 123:1577–1581[Abstract/Free Full Text]
  24. Courtin F, Chantoux F, Gavaret JM, Toru-Delbauffe D, Jacquemin C, Pierre M 1989 Induction of type II 5'-deiodinase activity in cultured rat astroglial cells by 12-O-tetradecanoylphorbol-13-acetate: dependence on glucocorticoids. Endocrinology 125:1277–1281[Abstract/Free Full Text]
  25. Courtin F, Gavaret JM, Toru-Delbauffe D, Pierre M 1990 Induction of 5'-deiodinase activity in rat astroglial cells by acidic fibroblast growth factor. Brain Res Dev Brain Res 53:237–242[Medline]
  26. Saunier B, Pierre M, Jacquemin C, Courtin F 1993 Evidence for cAMP-independent thyrotropin effects on astroglial cells. Eur J Biochem 218:1091–1094[Medline]
  27. Leonard JL, Siegrist-Kaiser CA, Zuckerman CJ 1990 Regulation of type II iodothyronine 5'-deiodinase by thyroid hormone. Inhibition of actin polymerization blocks enzyme inactivation in cAMP-stimulated glial cells. J Biol Chem 265:940–946[Abstract/Free Full Text]
  28. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  29. Courtin F, Liva P, Gavaret JM, Toru-Delbauffe D, Pierre M 1991 Induction of 5-deiodinase activity in astroglial cells by 12-O-tetradecanoylphorbol 13-acetate and fibroblast growth factors. J Neurochem 56:1107–1113[CrossRef][Medline]
  30. Esfandiari A, Courtin F, Lennon AM, Gavaret JM, Pierre M 1992 Induction of type III deiodinase activity in astroglial cells by thyroid hormones. Endocrinology 131:1682–1688[Abstract/Free Full Text]
  31. St. Germain DL 1988 The effects and interactions of substrates, inhibitors, and the cellular thiol-disulfide balance on the regulation of type II iodothyronine 5'-deiodinase. Endocrinology 122:1860–1868[Abstract/Free Full Text]
  32. Steinsapir J, Harney J, Larsen PR 1998 Type 2 iodothyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes. J Clin Invest 102:1895–1899[Medline]
  33. Semenza GL 2004 Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda) 19:176–182[CrossRef][Medline]
  34. Vordermark D, Kraft P, Katzer A, Bolling T, Willner J, Flentje M 2005 Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}). Cancer Lett 230:122–133[CrossRef][Medline]
  35. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ 2001 Targeting of HIF-{alpha} to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472[Abstract/Free Full Text]
  36. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin Jr WG 2001 HIF{alpha} targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292:464–468[Abstract/Free Full Text]
  37. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ 2001 C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107:43–54[CrossRef][Medline]
  38. Selak MA, Armour SM, MacKenzie ED, Boulahbel H, Watson DG, Mansfield KD, Pan Y, Simon MC, Thompson CB, Gottlieb E 2005 Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-{alpha} prolyl hydroxylase. Cancer Cell 7:77–85[CrossRef][Medline]
  39. Safran M, Kaelin Jr WG 2003 HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest 111:779–783[CrossRef][Medline]
  40. Masson N, Ratcliffe PJ 2003 HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O(2) levels. J Cell Sci 116:3041–3049[Abstract/Free Full Text]
  41. Khan A, Ling ZC, Landau BR 1996 Quantifying the carboxylation of pyruvate in pancreatic islets. J Biol Chem 271:2539–2542[Abstract/Free Full Text]
  42. Minet E, Michel G, Mottet D, Raes M, Michiels C 2001 Transduction pathways involved in hypoxia-inducible factor-1 phosphorylation and activation. Free Radic Biol Med 31:847–855[CrossRef][Medline]
  43. Goyal P, Weissmann N, Grimminger F, Hegel C, Bader L, Rose F, Fink L, Ghofrani HA, Schermuly RT, Schmidt HH, Seeger W, Hanze J 2004 Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species. Free Radic Biol Med 36:1279–1288[CrossRef][Medline]
  44. Block K, Gorin Y, Hoover P, Williams P, Chelmicki T, Clark RA, Yoneda T, Abboud HE 2007 NAD(P)H oxidases regulate HIF-2{alpha} protein expression. J Biol Chem 282:8019–8026[Abstract/Free Full Text]
  45. Abramov AY, Jacobson J, Wientjes F, Hothersall J, Canevari L, Duchen MR 2005 Expression and modulation of an NADPH oxidase in mammalian astrocytes. J Neurosci 25:9176–9184[Abstract/Free Full Text]
  46. Bianco AC, Larsen PR 2005 Cellular and structural biology of the deiodinases. Thyroid 15:777–786[CrossRef][Medline]
  47. Chavez JC, Baranova O, Lin J, Pichiule P 2006 The transcriptional activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes. J Neurosci 26:9471–9481[Abstract/Free Full Text]
  48. Coux O, Tanaka K, Goldberg AL 1996 Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65:801–847[CrossRef][Medline]
  49. Breitbart H 1981 Effect of ionophores and metabolic inhibitors on protein synthesis in rabbit reticulocytes. Biochim Biophys Acta 656:160–166[Medline]
  50. Niitsu Y, Hori O, Yamaguchi A, Bando Y, Ozawa K, Tamatani M, Ogawa S, Tohyama M 1999 Exposure of cultured primary rat astrocytes to hypoxia results in intracellular glucose depletion and induction of glycolytic enzymes. Brain Res Mol Brain Res 74:26–34[Medline]
  51. Kahlert S, Reiser G 2004 Glial perspectives of metabolic states during cerebral hypoxia—calcium regulation and metabolic energy. Cell Calcium 36:295–302[CrossRef][Medline]
  52. Lu H, Dalgard CL, Mohyeldin A, McFate T, Tait AS, Verma A 2005 Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1. J Biol Chem 280:41928–41939[Abstract/Free Full Text]
  53. Baqui MM, Gereben B, Harney JW, Larsen PR, Bianco AC 2000 Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy. Endocrinology 141:4309–4312[Abstract/Free Full Text]
  54. Courtin F, Pelletier G, Walker P 1985 Subcellular localization of thyroxine 5'-deiodinase activity in bovine anterior pituitary. Endocrinology 117:2527–2533[Abstract/Free Full Text]
  55. Steinsapir J, Bianco AC, Buettner C, Harney J, Larsen PR 2000 Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme’s active center. Endocrinology 141:1127–1135[Abstract/Free Full Text]
  56. Gereben B, Goncalves C, Harney JW, Larsen PR, Bianco AC 2000 Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation. Mol Endocrinol 14:1697–1708[Abstract/Free Full Text]
  57. Botero D, Gereben B, Goncalves C, De Jesus LA, Harney JW, Bianco AC 2002 Ubc6p and ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase. Mol Endocrinol 16:1999–2007[Abstract/Free Full Text]
  58. Kim BW, Zavacki AM, Curcio-Morelli C, Dentice M, Harney JW, Larsen PR, Bianco AC 2003 Endoplasmic reticulum-associated degradation of the human type 2 iodothyronine deiodinase (D2) is mediated via an association between mammalian UBC7 and the carboxyl region of D2. Mol Endocrinol 17:2603–2612[Abstract/Free Full Text]
  59. Weissman AM 2001 Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2:169–178[CrossRef][Medline]
  60. Dentice M, Bandyopadhyay A, Gereben B, Callebaut I, Christoffolete MA, Kim BW, Nissim S, Mornon JP, Zavacki AM, Zeold A, Capelo LP, Curcio-Morelli C, Ribeiro R, Harney JW, Tabin CJ, Bianco AC 2005 The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate. Nat Cell Biol 7:698–705[CrossRef][Medline]
  61. Zeold A, Pormuller L, Dentice M, Harney JW, Curcio-Morelli C, Tente SM, Bianco AC, Gereben B 2006 Metabolic instability of type 2 deiodinase is transferable to stable proteins independently of subcellular localization. J Biol Chem 281:31538–31543[Abstract/Free Full Text]
  62. Curcio-Morelli C, Zavacki AM, Christofollete M, Gereben B, de Freitas BC, Harney JW, Li Z, Wu G, Bianco AC 2003 Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau protein-interacting deubiquitinating enzymes regulates thyroid hormone activation. J Clin Invest 112:189–196[CrossRef][Medline]
  63. Li Z, Wang D, Na X, Schoen SR, Messing EM, Wu G 2002 Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor. Biochem Biophys Res Commun 294:700–709[CrossRef][Medline]
  64. Li Z, Na X, Wang D, Schoen SR, Messing EM, Wu G 2002 Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein. J Biol Chem 277:4656–4662[Abstract/Free Full Text]
  65. Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM 2006 Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1{alpha}, HIF-2{alpha}, and other pathways. J Biol Chem 281:15215–15226[Abstract/Free Full Text]
  66. Cockman ME, Lancaster DE, Stolze IP, Hewitson KS, McDonough MA, Coleman ML, Coles CH, Yu X, Hay RT, Ley SC, Pugh CW, Oldham NJ, Masson N, Schofield CJ, Ratcliffe PJ 2006 Posttranslational hydroxylation of ankyrin repeats in I{kappa}B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc Natl Acad Sci USA 103:14767–14772[Abstract/Free Full Text]
  67. Mottet D, Dumont V, Deccache Y, Demazy C, Ninane N, Raes M, Michiels C 2003 Regulation of hypoxia-inducible factor-1{alpha} protein level during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3ß pathway in HepG2 cells. J Biol Chem 278:31277–31285[Abstract/Free Full Text]
  68. Emerling BM, Platanias LC, Black E, Nebreda AR, Davis RJ, Chandel NS 2005 Mitochondrial reactive oxygen species activation of p38 mitogen-activated protein kinase is required for hypoxia signaling. Mol Cell Biol 25:4853–4862[Abstract/Free Full Text]
  69. Gleadle JM, Ebert BL, Ratcliffe PJ 1995 Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing. Eur J Biochem 234:92–99[Medline]
  70. Cotgreave I, Moldeus P, Schuppe I 1991 The metabolism of N-acetylcysteine by human endothelial cells. Biochem Pharmacol 42:13–16[CrossRef][Medline]
  71. Drost E, Lannan S, Bridgeman MM, Brown D, Selby C, Donaldson K, MacNee W 1991 Lack of effect of N-acetylcysteine on the release of oxygen radicals from neutrophils and alveolar macrophages. Eur Respir J 4:723–729[Abstract]
  72. Failli P, Palmieri L, D’Alfonso C, Giovannelli L, Generini S, Rosso AD, Pignone A, Stanflin N, Orsi S, Zilletti L, Matucci-Cerinic M 2002 Effect of N-acetyl-L-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis. Nitric Oxide 7:277–282[CrossRef][Medline]
  73. Hagen T, Taylor CT, Lam F, Moncada S 2003 Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1{alpha}. Science 302:1975–1978[Abstract/Free Full Text]
  74. Almeida A, Delgado-Esteban M, Bolanos JP, Medina JM 2002 Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture. J Neurochem 81:207–217[CrossRef][Medline]
  75. Rahman B, Kussmaul L, Hamprecht B, Dringen R 2000 Glycogen is mobilized during the disposal of peroxides by cultured astroglial cells from rat brain. Neurosci Lett 290:169–172[CrossRef][Medline]
  76. Heuer H, Maier MK, Iden S, Mittag J, Friesema EC, Visser TJ, Bauer K 2005 The monocarboxylate transporter 8 linked to human psychomotor retardation is highly expressed in thyroid hormone-sensitive neuron populations. Endocrinology 146:1701–1706[Abstract/Free Full Text]



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