CCAAT/Enhancer-Binding Protein-Homologous Protein Expression and Transcriptional Activity Are Regulated by 3',5'-Cyclic Adenosine Monophosphate in Thyroid Cells
Martine Pomerance,
Daniel Carapau,
Françoise Chantoux,
Michaël Mockey,
Claude Correze,
Jacques Francon and
Jean-Paul Blondeau
Unité 486, Institut National de la Santé et de la Recherche Médicale-Paris XI, Transduction Hormonale et Régulation Cellulaire, Faculté de Pharmacie, 92296 Châtenay-Malabry, France
Address all correspondence and requests for reprints to: Dr. M. Pomerance, Unité 486, Institut National de la Santé et de la Recherche Médicale-Paris XI, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cédex, France. E-mail: martine.pomerance{at}cep.u-psud.fr.
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ABSTRACT
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The cAMP pathway activates p38-MAPKs in the FRTL-5 rat thyroid cell line, contributing to the increased expression of the Na+/I- symporter (NIS) mRNA. This study investigates the cAMP-dependent expression and transcriptional activity of the p38-MAPK substrate CCAAT/enhancer-binding protein-homologous protein (CHOP). CHOP is expressed in the rat thyroid gland and in confluent PCCL3 and FRTL-5 cells. In FRTL-5 cells, TSH withdrawal induced a rapid down-regulation of CHOP that could be prevented by forskolin (Fk). Moreover, TSH and Fk were able to reinduce CHOP expression. The use of pharmacological inhibitors indicated that cAMP-induced CHOP expression was dependent on protein kinase A (PKA), mammalian target of rapamycin pathway, and reactive oxygen species. Transfection of a CHOP trans- reporting system revealed strong stimulation of the transcriptional activity of CHOP by Fk, by chlorophenylthio-cAMP, and by the catalytic subunit of PKA. CHOP transcriptional activity was significantly reduced by the p38-MAPK inhibitor SB203580, by transfection of a dominant-negative variant of p38
-MAPK, or by mutation of two serine residues in CHOP targeted by p38-MAPKs. Finally, cAMP-induced NIS mRNA expression was higher in FRTL-5 cells stably transfected with CHOP cDNA than in control cells. Likewise, the activity of the NIS promoter was higher in cells overexpressing CHOP than in control cells. These findings suggest that the stimulation of CHOP expression and transcriptional activity by the cAMP pathway may contribute to the regulation of genes involved in thyroid cell differentiation.
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INTRODUCTION
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TSH REGULATES THYROCYTE function, differentiation and proliferation. cAMP mediates most of the stimulant effects of TSH within the thyroid gland by coupling the TSH receptor to protein Gs and subsequently activating adenylyl cyclase. Thyrocyte proliferation is also stimulated by other factors (insulin, growth factors, cytokines, phorbol ester), which act via signaling cascades involving the MAPKs: ERKs, c-Jun N-terminal kinases (JNKs), and p38-MAPKs (1). Insulin and IGF-I also act synergistically with TSH to regulate the expression of differentiation markers of the thyroid cells (2).
The cAMP pathway was not able to induce an increase in the phosphorylation of ERKs and JNKs or of stimulating their kinase activity in CHO cells stably transfected with the human TSH receptor and in the FRTL-5 cell line (3). In contrast, the phosphorylation and the activity of p38-MAPKs were stimulated by TSH (4). This effect was also produced by forskolin (Fk), and by a permeant analog of cAMP, indicating that this was a cAMP-dependent effect. TSH also stimulated the MAPK kinases MKK3 and MKK6, which are activators of p38-MAPKs. p38-MAPK phosphorylation involved protein kinase A (PKA), the G protein Rac1, and generation of reactive oxygen species (ROS). Finally, TSH and Fk both stimulated the activity of MAPK-activated protein kinase-2, a known physiological substrate of p38-MAPKs.
The activation of the p38-MAPKs is usually associated with the responses to environmental stress and to proinflammatory cytokines such as IL-1 and TNF-
. However, recent studies have shown that the p38-MAPKs are also involved in regulating other functions, including the differentiation and the proliferation of some types of cells (5). p38-MAPKs are involved in the induction of the differentiation of various cell types, such as the chondrocyte and myocyte stem cells, and in the growth of neurites (6, 7, 8). We have reported that inhibition of the p38-MAPKs by SB203580 leads to a significant reduction in the expression of the Na+/I- symporter (NIS) mRNA stimulated by TSH or by Fk (4).
Several physiologically relevant transcription factors that are activated by p38-MAPKs have been identified in various cell types. p38-MAPKs can phosphorylate and activate the transcriptional function of these factors, either directly or via their protein kinase substrates MAPK-activated protein kinase-2/3. They include the transcription factors ATF-2 (9, 10), the ternary complex factors Elk-1 and Sap1a (11, 12), cAMP response element binding protein (CREB)/ATF-1 (13), serum response factor (14), p53 (15), myocyte enhancer factor-2C and -2A (16, 17). Members of the C/EBP (CCAAT/enhancer-binding protein) family of transcription factors, such as C/EBPß and CHOP (C/EBP-homologous protein), are also under the control of p38-MAPKs that directly phosphorylate and activate them (18, 19). However, nothing is known about the transcription factors targeted by p38-MAPKs within thyroid cells, except that the factors CREB and ATF1 are not activated by these MAPKs in response to stimulation by Fk (4).
In the present study, we investigated the expression in thyroid cells of the p38-MAPK model substrate CHOP and the regulation of its transcriptional activity by cAMP. CHOP was first identified as the product of a gene whose transcription is induced by cellular growth arrest and by genotoxic drugs (20, 21). For this reason, the gene is also known as GADD153 (growth arrest and DNA damage-inducible gene 153). It is also known as C/EBP
, due to the marked sequence identity of its carboxy-terminal basic-leucine zipper (bZIP) domain with that of other members of the C/EBP family (22). Several studies have reported a relationship between CHOP expression in cultured cells and various stressful conditions, such as the depletion of Ca2+ stores (23), oxidative stress (24), and glucose or amino acid deprivation (25, 26). CHOP has been reported to be involved in the apoptosis induced by endoplasmic reticulum stress (27), alkylating agents (19), or by Fas and ceramides (28), although the molecular mechanisms linking CHOP to the induction of apoptosis have not yet been elucidated. In contrast, CHOP expression increases the antiapoptotic effect of GH in mammary carcinoma cells (29). CHOP is also involved in regulating cell differentiation, either negatively as in adipocytes (30) or positively as in keratinocytes (31) or erythroid cells (32). CHOP can form heterodimers with other members of the C/EBP family, regulating gene expression either as a dominant-negative factor preventing the binding of the C/EBPs to their canonical DNA targets (33) or as a transcriptional activator by directing CHOP-C/EBP heterodimers to other DNA sequences (34).
Here, we found that CHOP was expressed in the rat thyroid gland and in PCCL3 and FRTL-5 rat thyroid cell lines when cells reached confluency, and that the regulation of its expression involved the cAMP pathway, ROS and the mammalian target of rapamycin (mTOR) pathway. The transcriptional activity of CHOP was also strongly stimulated by agents that elevate cellular cAMP and by transfection of the catalytic subunit of PKA, and this effect was partly dependent on p38-MAPK activity. FRTL-5 cells stably transfected with CHOP cDNA expressed higher levels of NIS mRNA in response to Fk than mock-transfected cells. These results show that CHOP expression and CHOP transcriptional activity are regulated by cAMP-dependent pathways involving in part p38-MAPK and suggest that CHOP contributes to the regulation of thyroid cell differentiation.
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RESULTS
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Inducible CHOP Expression in Rat Thyroid Cells
Western blot analysis showed that CHOP, corresponding to a 3032 kDa polypeptide, is expressed in the rat thyroid gland as well as in the rat thyroid cell lines PCCL3 and FRTL-5 (Fig. 1A
). The expression of CHOP was related to cell density in FRTL-5 cells cultured in Coons standard medium (Fig. 1B
). CHOP was barely detectable in growing cells but was markedly induced in confluent, growth-arrested cultures. To check that CHOP induction was not due to the development of adverse culture conditions, such as nutrient depletion, the cells were fed with fresh medium at frequent intervals. This did not change the level of CHOP expression at confluency (not shown).

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Fig. 1. CHOP Is Expressed in Rat Thyroid Gland and in Thyroid Cell Lines
FRTL-5 and PCCL3 cells were cultured in standard medium containing 5% calf serum, 0.5 mU/ml TSH, 10 µg/ml insulin, and 5 µg/ml transferrin and treated as described below. Thyroid glands were obtained from adult female Wistar rats. Total protein from cells and soluble protein from thyroid glands were extracted, quantified by amidoschwarz assay, and equal amounts of protein in each lane were analyzed by Western blot as described in Materials and Methods. A portion of the blots stained with Ponceau S is shown as a loading control (lower panels). A, Rat thyroid gland, confluent PCCL3 and FRTL-5 cells. B, FRTL-5 cells were cultured in standard medium to mid- or full confluency. C, Confluent FRTL-5 cells were incubated for the indicated times in deprivation medium lacking TSH, insulin and serum. D, Confluent FRTL-5 cells were either not deprived, or incubated for 48 h in deprivation medium containing: no addition (0), 10 µM Fk, 10 µg/ml insulin (Ins), 10 µM Fk plus 10 µg/ml Ins, 5% calf serum (S). The results are representative of at least two independent experiments.
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When confluent FRTL-5 cells were cultured in Coons deprivation medium (lacking TSH, insulin and serum), the amount of CHOP decreased rapidly and became very low within 624 h (Fig. 1C
). The decrease of CHOP expression could be prevented during a 48-h period by adding Fk to the Coons deprivation medium, at the time of deprivation, but not by adding insulin alone or serum. CHOP was expressed at even higher levels when Fk and insulin were simultaneously present in the Coons deprivation medium (Fig. 1D
).
To further assess the effect of TSH and cAMP on CHOP expression, confluent FRTL-5 cells were cultured in Coons deprivation medium to suppress CHOP expression. To study the effect of TSH, cells were deprived for 5 d (to allow TSH receptor reexpression after initial desensitization of the receptor) and then stimulated for different periods of time. Western blot analysis showed that TSH reinduced CHOP within 1538 h of treatment (Fig. 2A
, left panel). The effect of Fk on CHOP expression was studied after a 48-h deprivation period. Its effect was similar to that of TSH, whereas dideoxyforskolin (ddFk), a structural analog of Fk that does not activate adenylyl cyclase, had no effect (Fig. 2A
, right panel). The effect of insulin and Fk, alone or in association, on CHOP reinduction is shown in Fig. 2B
. Scanning densitometry of the bands (using NIH Image 1.60) in three independent experiments indicated that insulin was approximately four times less effective than Fk, and approximately 10 times less effective than Fk plus insulin after a 48-h stimulation period. This suggests that the effects of insulin and Fk were more than additive.

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Fig. 2. CHOP Expression Is Induced by TSH and Fk
FRTL-5 cells were cultured to confluency in standard medium and incubated in deprivation medium lacking TSH, insulin and serum. A, Cells were stimulated by TSH (1 mU/ml) for the indicated times after a 5-d deprivation period (left panel), or cells were treated for 38 h with vehicle (Ctrl), 10 µM Fk, or 10 µM ddFk after a 48-h deprivation period (right panel). B, Cells were stimulated by 10 µg/ml insulin (INS), or 10 µM Fk, or both (Fk + INS), for the indicated times after a 48-h deprivation period. Total protein was extracted, quantified by amidoschwarz assay, and equal amounts of protein were analyzed by Western blots for CHOP expression. A portion of the blots stained with Ponceau S is shown as a loading control (lower panels). Three separate experiments were performed with similar results.
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Northern blot analysis using a specific cDNA probe (Fig. 3
) showed that TSH stimulated the expression of CHOP mRNA with a time-course similar to that of CHOP protein induction, with a maximal level reached after 2440 h.

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Fig. 3. Induction of CHOP mRNA Expression by TSH
FRTL-5 cells were cultured to confluency in standard medium, incubated for 5 d in deprivation medium, and treated with TSH (1 mU/ml) for the times indicated. Total RNA was analyzed by Northern blot and CHOP mRNA was detected by hybridization to a specific 32P-labeled cDNA probe and quantitative autoradiography. The corresponding methylene-bluestained 18S rRNA bands are shown as loading controls (lower panel).
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Effects of Signal Transduction Inhibitors on CHOP Expression
To determine the possible involvement of PKA in cAMP-induced CHOP expression, FRTL-5 cells, previously cultured for 48 h in deprivation medium, were incubated without or with the PKA inhibitor H89 for 1 h and then stimulated by Fk for 38 h. Western blot analysis shows that the induction of CHOP expression by Fk was inhibited by 5 and 10 µM H89 (Fig. 4A
, left panel). Similarly, a 2-h preincubation with 100 µM of the cell-permeant protein kinase A inhibitor (PKI) resulted in an almost complete inhibition of Fk-induced CHOP expression (Fig. 4A
, right panel). Furthermore, 8-CPT-2'-O-Me-cAMP, a potent Epac activator (35), did not induce CHOP expression during a 38-h period, even at 100 µM (not shown). These results indicate that PKA is involved in the induction of CHOP expression by cAMP.

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Fig. 4. Effect of PKA Inhibitors and Antioxidants on cAMP-Induced CHOP Expression
FRTL-5 cells were cultured to confluency in standard medium and incubated for 48 h in deprivation medium. A, cells were either harvested as such (0) or pretreated for 1 h with increasing concentrations of H89 (left panel) or with 100 µM PKI (right panel) and further treated for 38 h without or with 10 µM Fk. B, Cells were either not pretreated (0) or pretreated for 1 h with 100 µM ascorbic acid (Asc), 20 mM N-acetylcysteine (NAC), or 550 U/ml catalase (Cat), and further stimulated, or not, for 38 h by Fk (10 µM). Total protein was extracted and equal amounts of protein were analyzed by Western blots for CHOP expression. A portion of the blots stained with Ponceau S is shown as a loading control (lower panels). The data shown are representative of two independent experiments.
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Thyroid cells respond to TSH by generating hydrogen peroxide (36), which acts as an electron acceptor in the oxidative reactions (iodination and coupling) involved in producing the thyroid hormones. On the other hand, the CHOP gene has been shown to be induced by H2O2 in HeLa cells (37). Therefore, deprived FRTL-5 cells were stimulated by Fk for 38 h in the presence of the antioxidants ascorbic acid, N-acetylcysteine or catalase. Ascorbic acid is a free radical scavenger, catalase removes extracellular H2O2, and N-acetylcysteine replenishes intracellular reduced glutathione stores. CHOP expression was then analyzed by Western blot (Fig. 4B
). N-acetylcysteine (20 mM) completely inhibited the Fk-induced CHOP expression. In contrast, ascorbic acid (100 mM) and catalase (550 U/ml) had no effect.
It was reported that the mTOR signaling pathway is involved in CHOP induction by H2O2 in mouse fibroblasts (38). We therefore investigated the effect of a specific inhibitor of the mTOR protein kinase on Fk-induced CHOP expression. Figure 5A
shows that rapamycin (20 nM) abolished the effect of Fk on CHOP expression in FRTL-5 cells. As a control, we observed that the same amount of rapamycin also blocked insulin-induced phosphorylation of p70-S6 kinase (S6K1) (Fig. 5B
). In contrast, the phosphatidylinositol-3 kinase (PI-3 kinase) inhibitor wortmannin, the MKK1/2 inhibitor PD98059 and the p38-MAPK inhibitor SB 203580 were without effect on CHOP expression (Fig. 5A
). Controls showing the activity of these inhibitors are shown in panels C to E.

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Fig. 5. Effect of Signal Transduction Inhibitors on cAMP-Induced CHOP Expression
FRTL-5 cells were cultured to confluency in standard medium and incubated for 48 h in deprivation medium. A, Cells were pretreated for 1 h in the absence (0) or presence of 50 µM PD98059 (PD), 10 µM SB 203580 (SB), 20 nM rapamycin (Rapa), or 100 nM wortmannin (W) and further stimulated, or not, for 38 h by Fk (10 µM). Total protein was extracted and equal amounts of protein were analyzed by Western blot for CHOP expression. A portion of the blots stained with Ponceau S is shown as a loading control (lower panels). The data shown are representative of three independent experiments. BE, Cells were pretreated in the absence or presence of the transduction inhibitors, as described above, and further stimulated, or not, with 10 µg/ml insulin (BD) or 10 µM Fk (E). Total protein was extracted and equal amounts of protein were analyzed by Western blot using anti-phospho-S6K1 (B, D), anti-phospho-ERK1/2 (C), or anti-phospho-p38-MAPK (E) antibodies, as described in Materials and Methods.
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cAMP Pathway Induces CHOP Transcriptional Activity
To test the functional activation of CHOP (Fig. 6
), FRTL-5 cells were cotransfected with a 5X-Gal4-luciferase reporter construct and an expression vector containing a chimeric gene in which the activation domain of CHOP was ligated to the Gal4 DNA binding domain (Gal4-CHOP). As expected, luciferase activity was strongly increased (25 ± 7-fold, n = 3 independent experiments) by cotransfection of an expression vector encoding a constitutively activated mutant of MKK6, which is a p38-MAPK activator (9). A 1-h treatment with Fk (10 µM), followed by a 4-h deprivation period, stimulated the transcriptional activity of Gal4-CHOP by 14 ± 5-fold (n = 5). In contrast, ddFK did not promote the transcriptional activity of Gal4-CHOP. One experiment performed with a vector expressing solely the Gal4 DNA binding domain (instead of the Gal4-CHOP expression vector) indicated that both the basal and Fk-stimulated transcriptional activity were abolished, and confirmed that the CHOP transactivation domain was responsible for the observed transcriptional effect (not shown). The transcriptional activity of CHOP was also stimulated by chlorophenylthio-cAMP (CPT-cAMP), a permeant analog of cAMP (17 ± 1-fold, n = 3) and by cotransfection of a plasmid expressing the catalytic subunit of PKA (12 ± 5-fold, n = 5), suggesting the probable involvement of the classical cAMP/PKA pathway (Fig. 6B
).

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Fig. 6. CHOP Transcriptional Activity Is Stimulated by MKK6 and Is Dependent on Activation of the cAMP Pathway
Luciferase reporter plasmid (0.4 µg) was transfected into FRTL-5 cells with 4 ng of Gal4-CHOP expression plasmid per culture well for 24 h, as described in Materials and Methods. After a 24-h incubation in deprivation medium, cells were treated and cell extracts were assayed 5 h later for luciferase activity. The values are mean ± SE of the indicated number (n) of independent experiments, each performed in triplicate. They are expressed relative to the luciferase activity measured in untreated cells transfected with only the luciferase reporter and Gal4-CHOP expression plasmid, which was arbitrarily defined as 1 (Ctrl). A, Cells were either cotransfected with 50 ng of expression vector coding for constitutively active MKK6 Glu, or treated with 10 µM Fk or 10 µM ddFk. B, Transfected cells were treated with 10 µM Fk or 0.5 mM chlorophenylthio-cAMP (CPT cAMP) or cotransfected with 25 ng of expression vector coding for the PKA catalytic subunit (cPKA).
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p38-MAPK Is Involved in cAMP-Induced CHOP Transcriptional Activity
To assess the requirement for p38-MAPK in Fk-induced CHOP transcriptional activity, we examined the effect of SB203580, a specific inhibitor of p38 MAPK
and ß, on luciferase activity in Fk-treated FRTL-5 cells. As seen in Fig. 7A
, the drug inhibited luciferase activity in a dose-dependent manner. However, this inhibition was not complete, reaching a maximum inhibition level of 4050% at the highest concentration used (10 µM). Analysis of p38-MAPK-mediated CHOP transcriptional activity was further studied by cotransfection of an expression plasmid encoding a dominant-negative form of p38-MAPK (p38-MAPK-AGF, in which threonine and tyrosine residues in the phosphorylation site have been replaced by alanine and phenylalanine, respectively). Expression of this dominant-negative p38-MAPK resulted in marked inhibition of the Fk-induced CHOP transcriptional activity (Fig. 7B
) with a magnitude similar to that obtained by SB203580 treatment. Wang and Ron (19) reported that CHOP undergoes stress-inducible phosphorylation of two serine residues (78 and 81), leading to enhanced transcriptional activity of CHOP. We therefore mutated the corresponding serine residues to alanine residues in the Gal4-CHOP fusion protein. The level of Fk-induced transcription was reproducibly lowered by approximately 45% when using Gal4-CHOP Ala 78, 81 instead of wild-type Gal4-CHOP (Fig. 7B
).

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Fig. 7. Fk-Stimulated CHOP Transcriptional Activity Is Partly p38-MAPK Dependent
FRTL-5 cells were cotransfected with luciferase reporter and Gal4-CHOP expression plasmids and incubated in deprivation medium before being stimulated, or not, with 10 µM Fk for 1 h, rinsed and further incubated in the deprivation medium for 4 h. The values are expressed as the percentage of the luciferase activity measured in cells stimulated by Fk in the absence of other treatment or plasmid cotransfection. A, Cells were pretreated for 60 min with the indicated concentrations of SB 203580 and then stimulated with Fk. B, Cells were pretreated with 10 µM SB 203580 (SB), or cotransfected with 40 ng of expression vector coding for a dominant-negative mutant of p38 -MAPK (p38-AGF), or were transfected with 4 ng of a mutated Gal4-CHOP expression plasmid (CHOP A78, 81) as described in Materials and Methods, instead of the Gal4-CHOP expression plasmid. The values are mean ± SE of the indicated number (n) of independent experiments, each performed in triplicate. C, Western blot detection of Gal4-CHOP proteins: cells were transfected (or not) as described above, and treated with Fk (or not) for 1 h followed by a further 4 h of incubation without Fk as for the luciferase assays. Cell extracts were analyzed as described in Materials and Methods, using an anti-CHOP antibody.
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As the cytomegalovirus (CMV) promoter that drives the expression of the Gal4-CHOP transcripts is cAMP-responsive, we examined the expression level of the Gal4-CHOP proteins by Western blot, using anti-CHOP antibody, under the conditions of Fk stimulation used to measure CHOP transcriptional activity. Figure 7C
shows that Fk treatment of the transfected FRTL-5 cells had no detectable effect on the expression of the Gal4-CHOP or Gal4-CHOP Ala78, 81 proteins under these conditions. This was confirmed by transfection of a ß-galactosidase expression vector driven by the CMV promoter (pCMV.SPORT-ßgal) and measurement of ß-galactosidase activity, indicating that a 1-h treatment by Fk, followed by a further 4-h incubation without Fk, stimulated ß-galactosidase expression by no more than 1012% (not shown).
These results indicate that p38-MAPK activation and subsequent phosphorylation of the transactivation domain of CHOP were involved in Fk-induced increase of CHOP activity in FRTL-5 cells.
Overexpression of CHOP Increases NIS mRNA Expression and NIS Promoter Activity
CHOP was overexpressed as a result of stable transfection of CHOP cDNA in FRTL-5 cells to study its role in cAMP regulation of thyroid cell differentiation. FRTL-5 cells were transfected with an expression plasmid containing CHOP cDNA under the control of a CMV promoter and encoding neomycin resistance. Stably transfected cells were selected by G418 resistance (FRTL-CHOP) and six clones were randomly selected and amplified. For control purposes, FRTL-5 cells were transfected with the empty vector, and G418-selected cells were pooled (FRTL-Neo). Three clones (3, 4, 6) of confluent FRTL-CHOP cells displayed markedly enhanced CHOP expression, even when they were cultured in deprivation medium (to suppress endogenous CHOP expression), compared with the level of endogenous CHOP present in Fk-treated wild-type FRTL-5 cells, as demonstrated by Western blot analysis (Fig. 8A
). Scanning densitometry (two experiments), indicated that clones 3, 4, and 6 expressed CHOP, respectively, approximately four times, approximately four times, and approximately seven times more than Fk-treated wild-type FRTL-5 cells. CHOP, expressed from the cAMP-responsive CMV promoter, was further increased by Fk treatment, with a maximum stimulation at 615 h of treatment (Fig. 8B
).

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Fig. 8. NIS mRNA Expression Is Increased in FRTL-5 Cells Stably Transfected with CHOP
FRTL-5, FRTL-CHOP, and FRTL-Neo cells were cultured to confluency in standard medium and incubated for 48 h in deprivation medium lacking TSH, insulin and serum. A, Western blot analysis of total protein extracts from FRTL-5 cells further treated for 40 h with 10 µM Fk (Wt + Fk), from FRTL-Neo cells (Neo), and from six clones of FRTL-CHOP cells. B, Western blot analysis of total protein extracts from FRTL-CHOP clone 6 further stimulated with 10 µM Fk for the indicated times. C, FRTL-CHOP clone 6 (+) and FRTL-Neo (-) cells were treated with 10 µM Fk for the indicated times. Total RNA was analyzed by Northern blot and NIS mRNA was detected by hybridization to a specific 32P-labeled cDNA probe and quantitative autoradiography. The corresponding methylene-blue-stained 18S rRNA bands are shown as loading controls (lower panel).
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The responsiveness of FRTL-CHOP (clones 3 and 6) and FRTL-Neo cells to Fk was investigated by measuring the stimulation of NIS mRNA expression by Northern blot. Figure 8C
(upper panel) shows that NIS mRNA levels had increased sharply 15 h after adding Fk and reached a plateau after 24 h. This time course of induction was the same as that observed in experiments performed with untransfected, wild-type FRTL-5 cells (not shown). FRTL-CHOP cells expressed higher levels of NIS mRNA in response to Fk than FRTL-Neo cells, particularly during the acute phase of induction. In two independent experiments performed with clone 6, the level of NIS mRNA had increased 1.5- to 2.0-fold relative to control cells 15 h after Fk stimulation. Under the same conditions, clone 3 showed a 2.2-fold higher level of NIS mRNA than control cells (not shown).
Regulation of the transcription of the rat NIS gene is attributed to a proximal promoter (up to -500 bp from the translation initiation site) and to an upstream enhancer (-2495 to -2260 bp), which bind both known and unknown factors that confer thyroid-specific regulation and TSH/cAMP responsiveness (39, 40). To study the effect of CHOP expression on the NIS promoter, we used a luciferase reporter plasmid containing the promoter region of the NIS gene, 2.9-kb upstream of the translation initiation site (pNIS-Luc1). The reporter construct was transiently transfected into FRTL-5 cells together with the pcDNA3-CHOP expression vector or the empty vector, and cells were cultured 24 h in deprivation medium (to suppress endogenous CHOP expression) before treatment for 7 h with Fk or vehicle and quantification of luciferase activity. Both the basal and the Fk-stimulated activities were significantly increased in cells transfected with pcDNA3-CHOP relative to cells transfected with the empty vector (Fig. 9A
). Similar results were obtained with FRTL-CHOP cells that constitutively overexpress CHOP, relative to FRTL-Neo cells (Fig. 9B
). Three experiments performed with clone 3 showed the same pattern of activation, with no significant difference in the mean activation elicited by CHOP expression (not shown). These data indicate that CHOP expression contributed to the stimulation of the NIS promoter. CHOP might exert its effect(s) either inside or outside the upstream enhancer. To test this, we used a luciferase reporter plasmid driven by the 2.2-kb regulatory 5'-region of the NIS gene (pNIS-Luc4), therefore lacking the upstream enhancer. The Fk responsiveness of the reporter gene was almost suppressed (Fig. 9C
), as expected from published observations (39), but the effect of CHOP overexpression remained similar to that observed with pNIS-Luc1.

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Fig. 9. Effect of Transient and Stable Overexpression of CHOP on NIS Promoter Activity
A, FRTL-5 cells were transiently cotransfected with 0.8 µg/well of the pNIS-Luc1 luciferase reporter plasmid together with 25 ng/well of the pcDNA3-CHOP expression vector or of the empty pcDNA3 vector. B, FRTL-CHOP (clone 6) and FRTL-Neo cells were transiently transfected with pNIS-Luc1 (0.8 µg/well). C, FRTL-CHOP (clone 6) and FRTL-Neo cells were transiently transfected with pNIS-Luc1 (0.8 µg/well) or pNIS-Luc4 (0, 73 µg/well). Twenty-four hours later, cells were cultured for 24 h in deprivation medium, treated for 7 h with 10 µM Fk (+) or vehicle (-) and cell extracts were assayed for luciferase activity. Values are expressed relative to the luciferase activity measured in unstimulated cells transfected with empty pcDNA3 (A) or relative to the luciferase activity measured in unstimulated FRTL-Neo cells (B and C). Results of experiments shown in panels A and C are the mean of triplicates. Similar results were obtained in another independent experiment. Results of experiments shown in panel B are the mean from six independent experiments, each performed in triplicate. Statistical analysis: P < 0.05 relative to unstimulated (*) or to Fk-stimulated (**) cells.
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DISCUSSION
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In previous studies, we have suggested the existence of a signaling pathway for TSH and cAMP in FRTL-5 cells, involving the p38-MAPKs, and depending on PKA, Rac1, and ROS (4). This signaling pathway is involved in regulating the expression of the NIS mRNA. In the present study, we investigated the downstream steps of cAMP and p38-MAPK signal transduction in FRTL-5 cells. We found that cAMP stimulates the expression and the activity of CHOP, a transcription factor which is expressed in the rat thyroid gland. CHOP is a known target of p38-MAPKs, which phosphorylate the CHOP transactivation domain on two conserved serine residues and consequently stimulate transcriptional activity of CHOP (19).
CHOP expression is known to be associated with growth arrest in a variety of cell types. However, it is not clear whether CHOP up-regulation is a consequence of growth arrest itself or is due to stressful conditions that can induce this state, such as treatment with genotoxic drugs (20, 21), treatment with agents that adversely affect protein maturation in the endoplasmic reticulum (23), oxidative stress (24), or glucose or amino acid starvation (25, 26). In our experiments, CHOP induction in confluent FRTL-5 cells was not due to nutrient depletion of the culture medium or accumulation of toxic metabolites, because it was not prevented by frequent renewal of the medium. On the other hand, CHOP is not induced by cell confluency in all cell types: thus, it is not induced in confluent fibroblasts unless the cells are also submitted to chemical stress (26).
The expression of CHOP is known to be up-regulated by a number of agents that cause cellular stress, but also by various hormones and factors, depending on the cell type. Thus, GH, erythropoietin, and fibroblast growth factor-2 stimulate CHOP expression via diverse signaling pathways in mammary carcinoma cells, erythroid cells and endothelial cells, respectively (29, 32, 41). Furthermore, the lactogenic hormones insulin and progesterone stimulate CHOP expression in normal mammary epithelial cells (42). Here, we report hormonal regulation of CHOP expression via the cAMP pathway. Maintaining a high level of its expression in confluent FRTL-5 cells required sustained stimulation by cAMP. Serum and insulin were unable to prevent the disappearance of CHOP after the withdrawal of TSH. Similarly, CHOP was reinduced mainly by Fk, although a synergistic effect of insulin and Fk was observed, which is a general feature of biological responses of thyroid cells to these hormones (43). PKA was likely to be involved in the signaling pathway because the PKA inhibitors, H89 and PKI, inhibited Fk-induced CHOP expression, whereas stimulation of Epac by a specific cAMP analog (35) did not induce CHOP expression.
Because a variety of oxidants has been shown to induce CHOP mRNA in HeLa cells (24), and because TSH has been shown to use ROS as signaling intermediates in FRTL-5 cells (4), we studied the involvement of ROS in CHOP induction in the latter cell line. We used ascorbic acid as a free radical scavenger, catalase to remove extracellular H2O2, and N-acetylcysteine to replenish intracellular reduced glutathione stores that are used by the H2O2-removing enzyme glutathione peroxidase. We found that N-acetylcysteine was effective in inhibiting Fk-induced CHOP expression, whereas ascorbic acid and catalase were not. This suggests that intracellularly generated H2O2 may be the signaling species rather than free radical species, such as the superoxide radical, or than extracellularly produced H2O2.
At present, the detailed mechanism by which cAMP induces the expression of CHOP is not known. TSH up-regulation of CHOP mRNA paralleled that of the protein, but regulation may be transcriptional or posttranscriptional. There is a composite C/EBP-ATF/CREB binding site in the CHOP gene promoter that mediates the responses to metabolic stress or arsenite (44) and to amino acid starvation (45). This site binds members of the ATF/CREB family of transcription factors and therefore could be a potential target for cAMP regulation of CHOP transcription. In addition, an activator protein-1 binding site was found to play a role in CHOP induction by oxidant treatment (46). This site could be the target of H2O2 generated after Fk stimulation of FRTL-5 cells. We also observed that rapamycin suppressed the induction of CHOP by cAMP signaling. Rapamycin is an inhibitor of the protein kinase mTOR, which targets the translational machinery through 4EBP1 and p70-S6 kinase (47). TSH and 8-bromo-cAMP have been reported to activate p70-S6 kinase in the WRT rat thyroid cell line (48), and we have obtained similar results in FRTL-5 cells (Pomerance, M., unpublished results). It has been reported that in mouse fibroblasts, the induction of CHOP by H2O2 is mediated through the PI-3 kinase and mTOR signaling pathways (37), but neither TSH nor Fk stimulates PI-3 kinase activity in FRTL-5 cells cultured in the absence of insulin (4), and the PI-3 kinase inhibitor wortmannin was without effect on Fk-induced CHOP expression. The activity of mTOR might be required for the expression of a protein, such as a transcription factor, that activates the transcription of the CHOP gene.
Stimulation of adenylyl cyclase in thyroid FRTL-5 cells resulted not only in the up-regulation of CHOP expression, but also in an increase in CHOP trans-activation function. We used a CHOP-specific trans-activation reporter assay that was stimulated, as expected, by expression of an activated form of MKK6, which is a selective activator of p38-MAPK (9). We report here that Fk stimulation of FRTL-5 cells resulted in a marked increase in the transcriptional activity of CHOP. We have demonstrated that the cAMP pathway is responsible for this effect, because Fk can be replaced by a permeant analog of cAMP and by transfection of PKA, but not by ddFK, which does not activate adenylyl cyclase. To our knowledge, this is the first time that a stimulatory effect of cAMP on the activity of this transcription factor has been reported.
We also report that Fk-stimulated transcriptional activity of CHOP is partly p38-MAPK dependent. This is consistent with the demonstration that p38-MAPKs phosphorylate the transactivation domain of CHOP on two adjacent serine residues (S78 and S81), resulting in the enhanced transcriptional activity of CHOP (19). The involvement of p38-MAPK in the stimulation of CHOP transcriptional activity by GH has also been reported (49). Part of the Fk-stimulated transcriptional activity of CHOP was not inhibited by the p38
/ß-MAPK inhibitor, SB 203580, or by coexpression of a dominant-negative form of p38
-MAPK. Incomplete inhibition could be due to the involvement of other p38-MAPK isoform(s), such as SB 203580-insensitive p38
-MAPK, which is expressed in the thyroid (50), or to a p38-MAPK-independent mechanism. Mutation of the serine residues 78 and 81, that are targeted by p38-MAPK (19) in the CHOP transactivation domain, to alanine residues also promoted partial inhibition of Fk-induced transcriptional activity when the mutated Gal4-CHOP fusion protein was transfected in FRTL-5 cells. Wang et al. (51) also reported that p38-MAPK-dependent phosphorylation of CHOP on serine residues 78 and 81 plays a role in mediating gene induction by ER stress in mouse fibroblasts, but that part of the signal is not dependent on the phosphorylation of these serine residues. It is not known whether CHOP may be a direct target of PKA (although the transactivation domain of CHOP lacks a PKA consensus phosphorylation site) or whether a transcriptional coactivator is able to interact with the CHOP transactivation domain only after this partner has itself been phosphorylated by PKA.
The precise mechanisms of the actions of CHOP are still unknown, but a number of studies have revealed functional links between CHOP expression and modifications of cell functions such as growth arrest and apoptosis, but also differentiation or protection from apoptosis. To investigate the modifications of cell functions induced by CHOP in FRTL-5 cells, we established FRTL-5-derived cell lines stably transfected with a rat CHOP expression vector (FRTL-CHOP cells). These cell lines did not undergo growth arrest or apoptosis under standard culture conditions. This may suggest that an additional proapoptotic signal would be necessary to induce apoptosis in FRTL-CHOP cells. It is unlikely that a survival signal, such as activation of the cAMP pathway, may counteract the proapoptotic effect of CHOP because FRTL-CHOP cells were able to survive in serum- and hormone-depleted medium.
Iodide transport into the thyroid cells is the main regulatory step in thyroid hormone biosynthesis. It is performed by NIS, the expression of which is regulated by TSH (52). FRTL-CHOP cells consistently expressed higher levels of NIS mRNA than control cells in response to adenylyl cyclase stimulation, pointing to an involvement of CHOP in FRTL-5 cell differentiation. Results obtained with a luciferase reporter driven by regulatory 5'-regions of the rat NIS gene are compatible with a cooperative transcriptional effect of CHOP and Fk on NIS mRNA expression. The upstream enhancer region (-2495 to -2260 from the translation initiation site) that confer cAMP responsiveness does not seem to be essential for CHOP stimulation. CHOP may exert its regulatory effects either directly, or indirectly by modulating the activity of other transcription factors. CHOP does not form homodimers but can heterodimerize with other bZIP transcription factors. Consequently, it can act as a dominant-negative regulator of the activity of C/EBP transcription factors (33) or it can activate transcription by interacting with C/EBPß or activator protein-1 factors (34, 46). The latter mechanism could be involved in up-regulating NIS transcription, but CHOP could as well counteract the effect of a negative regulator of NIS transcription.
In conclusion, CHOP expression is up-regulated by TSH and by the cAMP-elevating agent Fk through activation of PKA. CHOP transcriptional activity is also stimulated by cAMP via pathways involving in part p38-MAPK. Consistent with its role as a transcription factor, CHOP is involved in stimulating NIS promoter activity in FRTL-5 cells, resulting in the increased expression of NIS mRNA. These findings suggest that stimulation of CHOP expression and of its transactivating functions may play a significant role in the regulation of genes involved in thyroid cell growth and differentiation.
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MATERIALS AND METHODS
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Cell Cultures
The rat thyroid cell lines, FRTL-5 and PCCL3, were gifts from Dr. M. Eggo (Birmingham, UK) and Pr. R. Di Lauro (Napoli, Italy). They were grown to confluence in standard medium composed of Coons modified Hams F-12 medium (Seromed, Strasbourg, France) supplemented with 5% heat-inactivated new born calf serum (Life Technologies, Gaithersburg, MD), 0.5 mU/ml bovine TSH (Sigma, St. Louis, MO), 10 µg/ml insulin (Sigma) and 5 µg/ml transferrin (Sigma), 2 mM GlutaMAX (Life Technologies) in a 37 C, 5% CO2 incubator. In some experiments, serum and hormones were withdrawn (deprivation medium) from confluent cultures 48 h or 5 d before stimulation. Stably transfected FRTL-5 cells were cultured under the same conditions in the presence of 0.2 mg/ml geneticin (G418).
Plasmids and Plasmid Constructions
The fusion Gal4-CHOP trans-activator plasmid (pFA-CHOP) and the luciferase reporter plasmid (pFR-Luc) were purchased from Stratagene (La Jolla, CA). pFC2-dbd plasmid (Stratagene) was the negative control for the pFA plasmid used to check that the observed effects were not due to the Gal4 DNA binding domain. pFC-PKA (driven by a CMV promoter) was from Stratagene. pCMV.SPORT-ßgal was from Invitrogen Corp. (Carlsbad, CA). The QuikChange site-directed mutagenesis kit (Stratagene) was used to mutate two serine residues within the transactivation domain of the CHOP-Gal4 fusion protein (S79A, S82A). The sense oligonucleotide used was 5'-AGCACCTCCCAAGCCCCTCGCGCTCCAGATTCC (the mutated nucleotides are underlined). The PCR conditions used were those recommended by the manufacturer, and the resulting clones were sequenced to confirm the mutations. To generate a rat CHOP expression vector, CHOP ORF was PCR-amplified from rat thyroid cDNAs with a forward primer containing the BamHI site (5'-CGGGATCCATGGCAGCTGAGTCTCTG) and a reverse primer containing the EcoRI site (5'-CGGAATTCATGCTTGGTGCAGACT). The PCR-amplified CHOP cDNA was ligated to pcDNA3 in BamHI/EcoRI site to obtain the pcDNA3-CHOP expression plasmid. The resulting construct was cloned and sequenced. Constitutively active MKK6 (pcDNA3-MKK6 Glu) and dominant-negative p38
MAPK (pCMV5-p38-AGF) were gifts from Dr. J. Raingeaud (9). The luciferase reporter genes controlled by either the 2.9-kb region (pNIS-Luc1) or the 2.2-kb region (pNIS-Luc4) 5' to the rat NIS gene were gifts from Pr. R. Di Lauro (39).
Generation of FRTL-5 Cells Stably Transfected with Rat CHOP cDNA
FRTL-5 cells grown in 50-cm2 flasks under standard conditions were transfected with 3 µg of either the rat CHOP cDNA expression plasmid or the corresponding empty vector (pcDNA3), using Fugene reagent (Roche Molecular Biochemicals, Indianapolis, IN). Stable transfectants were selected with 0.2 mg/ml G418 for 2 wk. Six clones of FRTL-5 cells transfected with CHOP cDNA were amplified and designated FRTL-CHOP (clones 16), whereas the pool of vector-transfected control cells were designated FRTL-Neo. The expression of CHOP was subsequently determined by Western blot analysis.
Transient Transfection Procedure and Luciferase Assays
One day before being transfected, wild-type FRTL-5, FRTL-CHOP or FRTL-Neo cells were seeded to approximately 70% confluence in six-well plates. For studies of CHOP-mediated transcriptional activation in FRTL-5 cells, 0.4 µg of pFR-Luc reporter plasmid was cotransfected with 4 ng of the fusion trans-activator plasmid (pFA-CHOP or pFC2-dbd) with or without other expression plasmids or their empty counterparts. Transient transfections were performed using Fugene according to the manufacturers protocols. After incubating for 24 h, cells were placed in Coons deprivation medium without hormones and serum for an additional 24-h period. Cells were treated with Fk or the vehicle (Me2SO) for 1 h, rinsed and further incubated in the deprivation medium for 4 h. For studies of NIS promoter activity, FRTL-CHOP and FRTL-Neo cells were cotransfected using Fugene with 0.8 µg pNIS-Luc1 or 0.73 µg pNIS-Luc4, and 10 ng pCMV.SPORT-ßgal, whereas wild-type FRTL-5 cells were additionally cotransfected with 25 ng pcDNA3-CHOP (or 25 ng empty pcDNA3 as a control). Twenty-four hours after transfection, cells were transferred into deprivation medium for an additional 24-h period. The cells were stimulated for 7 h with Fk (or vehicle). Luciferase activities were measured using a luciferase reporter gene assay kit (Roche Molecular Biochemicals) according to the manufacturers protocol. They were normalized on the basis of protein content (DC Protein assay, Bio-Rad Laboratories, Hercules, CA). In experiments performed to study NIS promoter activities, luciferase values obtained for a given treatment (Fk or Me2SO) were also normalized for ß-galactosidase activities to correct for differences in transfection efficiencies between the different cell types or clones used. All transfections were run in triplicate, and each set of experiments was repeated several times, as reported in the text. Values represent the mean ± SE from multiple independent experiments or from triplicates, as mentioned in the figure legends.
Western Blot Analysis
Total lysates from PCCL3 or FRTL-5 cells were prepared by scraping the cells into Laemmli sample buffer. Thyroid glands from adult female Wistar rats were homogenized in extraction buffer A [10 mM Tris-HCl (pH 7.5), 420 mM NaCl, 10 mM EDTA, 1% NP40, 1% sodium deoxycholate, 0.05% SDS, 1 mM Na3VO4, 1 mM AEBSF, 1 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml antipain, 1 µg/ml pepstatin]. The homogenate was sonificated and the 100,000 x g supernatant was collected. Protein concentration was determined by the amidoschwarz assay (53). Proteins were resolved by 14% SDS-PAGE, transferred onto nitrocellulose membranes, and stained with Ponceau S. 3% nonfat dry milk-saturated membranes were probed overnight with 1 µg/ml anti-CHOP (F168) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The antibody binding was revealed using antirabbit IgG peroxidase conjugate antibody (Promega, Madison, WI) followed by the enhanced chemiluminescence kit (Amersham Biosciences, Saclay, France). For Western blot analysis of phospho-S6K1, phospho-ERK1/2, and phospho-p38-MAPK, cell extracts were obtained as described previously (4). Anti-phospho-S6K1 (Thr 389), anti-phospho-ERK1/2 (Thr 202/Tyr 204) and anti-phospho-p38-MAPK (Thr 180/Tyr 182) were purchased from New England Biolabs (Beverly, MA).
RNA Analysis
Total RNA from FRTL-5 cells was extracted using the RNABle kit (Eurobio, Les Ulis, France) according to the manufacturers protocol, and analyzed by Northern blot. The rat CHOP DNA probe was a BamHI/EcoRI restriction fragment obtained from the pCDNA3-CHOP plasmid. The rat NIS cDNA probe was isolated by RT-PCR using the following primers: sense (5'-CCTTCTGGACTTTCATAGTGGG) and antisense (5'-TGGGACCAGTAAGGTAGCTGAT) as previously described (4). Blots were stained with methylene-blue and hybridized with the probes labeled by random priming extension using the Megaprime labeling kit (Amersham Pharmacia Biotech) and [
32P]deoxy-CTP (3000 Ci/mmol) (New England Nuclear). Membranes were analyzed by quantitative autoradiography using an InstantImager (Packard, Canberra, Canada).
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ACKNOWLEDGMENTS
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We thank Dr. M. Eggo, Dr. J. Raingeaud, and Pr. R. Di Lauro for providing materials used in this study. We also acknowledge the excellent technical contribution of A. Dos Santos and M. Lévy.
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FOOTNOTES
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Abbreviations: ATF, Activating transcription factor; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP-homologous protein; CMV, cytomegalovirus; CPT-cAMP, chlorophenylthio-cAMP; CREB, cAMP-response element binding protein; ddFk, dideoxyforskolin; Fk, forskolin; MKK, MAPK kinase; mTOR, mammalian target of rapamycin; NIS, Na+/I- symporter; pFa, fusion Gal4-CHOP trans-activator; PI-3 kinase, phosphatidylinositol-3 kinase; PKA, protein kinase A; PKI, protein kinase A inhibitor; ROS, reactive oxygen species.
Received for publication December 3, 2002.
Accepted for publication August 1, 2003.
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