Cross-Talk between Signal Transducer and Activator of Transcription (Stat5) and Thyroid Hormone Receptor-ß 1 (TRß1) Signaling Pathways

H. Favre-Young, F. Dif, F. Roussille, B. A. Demeneix, P. A. Kelly, M. Edery and A. de Luze

INSERM Unité 344 (H.F.-Y., R.F., K.P., E.M.) Endocrinologie Moléculaire Faculté de Médecine Necker Paris cedex 15, France 75730
Laboratoire de Physiologie Générale et Comparée (FY.H., D.F., B.D., L.A.) Muséum National d’Histoire Naturelle UMR 8572 CNRS Paris cedex 05, France 75231


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL and T3 are involved in antagonistic regulations during various developmental processes in vertebrate species. We have studied cross-talk between transcription factors activated by these signaling pathways, i.e. signal transducer and activator of transcription 5 (Stat5) and thyroid hormone receptor ß1 (TRß1). Liganded TRß1 in the presence of its heterodimeric partner, retinoid X receptor {gamma} (RXR{gamma}), inhibited the PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60%. This T3-inhibitory effect studied on Stat5 activity was partly reversed by overexpression of a TRß1 dominant negative variant mutated within its nuclear localization signal (TR2A). We next showed that TRß1 and TR2A in the presence of RXR{gamma} increased and decreased, respectively, Stat5 localization into the nucleus regardless of hormonal stimulation. Thus, our data suggest that TRß1 can be associated with Stat5 in the cytoplasm and may be involved in Stat5 nuclear translocation. In PRL-treated cells overexpressing TRß1/RXR{gamma}, both Stat5 and TRß1 were coimmunoprecipitated, indicating physical association of the two transcription factors. In these cells, addition of T3 with ovine (o)PRL decreased the amounts of total and tyrosine-phosphorylated Stat5 in the cytoplasm compared with oPRL-treated cells. In the nucleus, no clear difference was observed on Stat5 DNA-binding after treatment with PRL and T3 vs. PRL alone in TRß1/RXR{gamma} transfected cells. However, antibodies directed against TRß1 lowered Stat5-DNA binding and addition of the deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5 transcriptional activity. Thus, we postulated that the negative cross-talk between TR and Stat5 on target genes could involve histone deacetylase recruitment by liganded TRß1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL plays a crucial role in several aspects of vertebrate physiology. PRL effects are related to growth and reproduction (1). In mammals, the effect of PRL on the mammary gland is well known, whereas in lower vertebrates its role is mainly in osmoregulation and metamorphosis (2). It is now well documented that the actions of PRL are mediated through the intracellular Janus kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5) signaling pathways (3). For the Stats transcription factors family, it is their tyrosine phosphorylation by Jak2 upon hormonal stimulation, which prompts Stat5 dimerization, nuclear import, DNA binding, and activation of target gene. Steroid/thyroid hormones have been frequently implicated in synergistic or antagonistic effects with both PRL and GH, the receptors of which are closely related and share similar signal transduction pathways (3, 5). In particular, thyroid hormone (TH) exerts antagonist effects on PRL action notably in mammary gland development or amphibian metamorphosis. Indeed, hyperthyroidism has been shown to suppress PRL-mediated mammary growth in virgin rats (6, 7), associated with increased lethality of the pups and absence of lactation after gestational delivery (8, 9, 10, 11, 12). Conversely, in amphibians, high levels of PRL have been shown to inhibit T3-induced metamorphosis (4, 13).

THs are implicated in numerous processes such as growth, maturation, development, and metabolism of most tissues in mammals (14, 15, 16). Addition of T3 leads to the activation of their nuclear receptors and modulation of target gene expression. Two main loci encoding thyroid hormone receptor {alpha} (TR{alpha}) and ß (TRß) isoforms exist, each isoform differing somewhat in structure (17). Based on hormone binding assays, it has been proposed that TRs are nuclear regardless of their ligand status (18). However, the precise distribution and the functional role of TR isoforms are not yet entirely understood. Indeed, different functional roles of TRß and TR{alpha} were first shown on a rat TRH transcript in vitro (19), then in vivo by somatic gene transfer (20). Such findings were confirmed with studies in knockout mice, in which TRß was shown to be dispensable for normal developmental expression of some T3-dependent target gene (21, 22, 23). Like many receptors belonging to the superfamily of steroid/thyroid nuclear receptors, thyroid hormone receptors (TRs) bind to specific TH-DNA responsive elements (TRE) upstream of target gene in heterodimeric complex with the 9-cis retinoid acid receptor (RXR) (24). Recently, a number of studies have shown a cross-talk between Stat5a and b with nuclear steroid receptors (25), such as glucocorticoid receptor (GR) (26, 27), estrogen receptor (ER) (27), progesterone receptor (PR) (28), and peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) (29). Unliganded PR or GR are essentially cytoplasmic. Thus, the observation that progestin or dexamethasone treatment induces Stat5 translocation into the nucleus, possibly mediated by the physical association of these liganded nuclear receptors with inactivated Stat5 (27, 28, 30), is most important. Nuclear entry of steroid nuclear receptors appears to be mediated by the interaction of nuclear localization signals (NLSs) within the proteins and specific NLS-binding proteins in nuclear pores (31, 32, 33). In contrast, no residue comprising a Stat NLS has been yet demonstrated, but dimerization of Stat5 molecules upon PRL stimulation has been postulated to unmask a NLS (34). Thus, the mechanisms underlying nuclear entry of numerous proteins, such as Stats monomers or homo- and heterodimers that are present in the cytoplasm, remain to be clarified.

In the present study, we investigated a possible inhibitory involvement of T3 in the oPRL-induced activation of a target gene in the HEK293 cell line. We carried out transfections so as to express elements of both the PRLR/Stat5a signaling pathway and the TRß1/RXR{gamma} pathway. We show that in TRß1/RXR{gamma}-transfected cells T3 markedly repressed oPRL-dependent Stat5a or Stat5b-induced reporter gene expression under the control of a lactogenic responsive element (LHRE) or the ß-casein promoter. We also show that overexpression of TRß1/RXR{gamma} results in an increased nuclear localization of Stat5a regardless of ligand stimulation. Conversely, overexpression of a dominant negative TRß1 mutated in its NLS decreases both Stat5a nuclear localization and T3-dependent inhibitory effects on PRL-induced transactivation of the LHRE-thymidine kinase (TK)-luciferase promoter. We also show the ability of Stat5a to associate with TRß1 in cytoplasmic extracts of oPRL and oPRL + T3 treated cells. Furthermore, in oPRL-treated cells, addition of T3 reduces phosphorylated Stat5a in the cytosol. In electrophoretic mobility shift assay (EMSA) studies, with LHRE probe, no band was supershifted after addition of antibodies directed against TRß1. However, TRß1 antibodies lowered Stat5a nuclear DNA binding when TRß1/RXR{gamma} was overexpressed. Finally, we show that addition of the histone deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5a transcriptional activity. Thus, our data indicate that TRß1/RXR{gamma} modifies the subcellular distribution of Stat5a in unstimulated as well as in stimulated cells. The inhibitory effect of T3 could result from an heterocomplex formation between Stat5a and recruitment by liganded TRß1 of a histone deacetylase within the transcriptional machinery in PRL-stimulated cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T3 in the Presence of TRß1 Alone or in Combination with RXR{gamma} Inhibits oPRL-Induced Transcriptional Activity
We have previously reported that heterologous cell systems, supplied with the gene encoding the PRLR and the ß-casein promoter-luciferase construct, can be used to study PRL-induced signaling and activation of transcription (35). In the present study, HEK293 cells were cotransfected with expression vectors encoding the PRLR, TRß1, and the LHRE-TK-luciferase reporter gene, with or without RXR{gamma}. Without RXR{gamma}, ovine (o)PRL treatment caused a 10-fold induction of luciferase expression from the LHRE (Fig. 1AGo, lane 2) as compared with controls (Fig. 1AGo, lane 1). T3 alone had no effect on luciferase expression compared with control. However, when T3 was added with oPRL, it inhibited LHRE-dependent luciferase expression, by up to 20%, as compared with oPRL-treated cells.



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Figure 1. T3 in the Presence of TRß1/RXR{gamma} Heterodimer Inhibits oPRL-Induced LHRE-TK-Luciferase Activity

A, Cells were transfected with TRß1, PRLR, LHRE-TK-luciferase, and the ß-galactosidase expression vectors. As indicated, cells were stimulated by oPRL (1 µg/ml), hGH (1 µg/ml), and/or T3 (10- 8 M) added during 10–12 h in the starvation medium (at 37 C). B, Cells were transfected with TRß1/RXR{gamma}, PRLR, LHRE-TK-luciferase, and ß-galactosidase. The same hormonal treatment was applied as described above. C, Cells were transfected with GHR, TRß1, LHRE-TK-luciferase, and ß-galactosidase expression vectors and stimulated as described above. Cells were treated as described previously. The relative light units were measured and normalized for ß-galactosidase activity. Results are the mean ± SE of three independent experiments, each performed in duplicate.

 
The same experimental procedure performed with RXR{gamma} did not modify the basal or the oPRL-stimulated LHRE-dependent luciferase expression compared with controls (Fig. 1BGo, lane 7). In contrast, addition of T3 in TRß1/RXR{gamma} transfected cells resulted in a higher level of inhibition (<60%) of oPRL-induced LHRE-dependent luciferase expression (Fig. 1BGo, lane 8). This T3-induced inhibition of the PRL transduction pathway was dose dependent in cells expressing either TRß1 alone or TRß1/RXR{gamma} heterodimers. Optimal inhibition occurred at 10-8 M T3 (range from 10-12 M to 10- 6 M, data not shown). As shown in Fig. 1CGo, a similar T3 inhibitory effect on Stat5-mediated LHRE-TK-luciferase activity was obtained when cells were transfected with GHR following the same experimental procedure.

We next examined whether this inhibitory effect of T3 on LHRE-induced reporter gene activity was specifically dependent on Stat5a or Stat5b (Fig. 2Go, A and B). For comparison, we studied the inhibitory effect of TRß1/RXR{gamma} on the wild-type ß-casein promoter linked to luciferase reporter gene activity (Fig. 2BGo). For this purpose, the same experimental procedure in the presence of TRß1/RXR{gamma} with PRLR was applied. As shown in Fig. 2Go, A and B, we obtained the same inhibitory effects after stimulation by oPRL + T3 in cells transiently cotransfected with the ß-casein promoter and Stat5a or Stat5b relative to those expressing LHRE (Fig. 2BGo). However, Stat5a and Stat5b differentially potentiated LHRE and ß-casein promoter activities. As shown in Fig. 2Go, comparison of Stat5a/Stat5b-mediated activation shows a more efficient transactivation potency of Stat5a than Stat5b on both the ß-casein promoter and the LHRE promoter. Thus, in our studies, Stat5a seems to be the member of Stat family involved as well as in LHRE or ß-casein promoter activities. Moreover, the higher sensitivity to Stat5a-mediated transcriptional activity was more apparent using LHRE-luciferase promoter than with the ß-casein promoter. Indeed, similar induction by oPRL was observed in control and cells overexpressing Stat5b with LHRE (Fig. 2AGo), whereas oPRL was unable to potentiate ß-casein promoter activity in control cells in the absence of added Stat5a or Stat5b (Fig. 2BGo).



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Figure 2. T3 Inhibits oPRL-Induced LHRE-TK-Luciferase and ß-Casein-Luciferase Activity in the Presence of TRß1/RXR{gamma}, Stat5a, or Stat5b

A, Cells were transfected with TRß1/RXR{gamma}, PRLR, LHRE-TK-luciferase, ß-galactosidase, and Stat5a or Stat5b. As indicated, hormonal treatment was performed in panels A and B as described in Fig. 1Go. B, Cells were transfected with same plasmids as in panel A but with ß-casein-TK-luciferase. Arbitrary units are calculated as a percent toward activity in oPRL-treated cells (lane 11). Results are the mean ± SE of three independent experiments, each performed in duplicate.

 
Effect of a NLS-Mutated TRß1 (GFP-TR2A) on LHRE-Luciferase Activity
To further examine the TRß1-dependent, T3-induced inhibition of Stat5a transcriptional activity, we used a dominant negative receptor with alanine substitution of Lys184-Arg185 on the NLS present in TRß1 (D-domain) (36). Transient transfections were performed using green fluorescent protein (GFP)-TRß1 or the NLS-mutated GFP-TR2A (Fig. 3Go). Luciferase expression estimated after cotransfection of GFP-TRß1 with RXR{gamma}, PRLR, and the LHRE-TK-luciferase in the presence of T3, oPRL, and T3 + oPRL showed similar fold-induction of luciferase activity, as previously observed with the wild-type TRß1 (compare Fig. 3AGo with Fig. 1BGo). However, transfection of the NLS-mutated GFP-TR2A sharply attenuated, from 60% to 20%, the T3-inhibitory effect on PRL-induced LHRE-TK-luciferase expression obtained with the wild-type GFP-TRß1 (Fig. 3BGo).



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Figure 3. Effect of T3 on LHRE-TK-Luciferase Activity in GFP-TRß1- and GFP-TR2A-Transfected Cells

Cells were transfected with PRLR, RXR{gamma}, and GFP-TRß1 (A) or GFP-TR2A (B). Cells were stimulated with T3, oPRL, or both as described in Fig. 1Go. The relative light units were measured and normalized for ß-galactosidase activity. Results are the mean ± SE of three independent experiments, each performed in duplicate.

 
Effect of TRß1/RXR{gamma} on Stat5a DNA Binding
The direct or indirect interactions between Stat5a and TRß1 were investigated using an electrophoretic mobility shift assay (EMSA) for Stat5a/LHRE protein-protein-DNA complex. The consensus LHRE DNA sequence from the ß-casein promoter gene was used in this experience. The 24 mers oligonucleotide sequence comprised one copy of the Stat5a response element (5'-TTTCTTGGAA-3'). Nuclear extracts used were from cells transfected with PRLR, Stat5a, TRß1, and RXR{gamma} (Fig. 4Go, A and C) or with PRLR and Stat5a only (Fig. 4BGo). Twenty four hours later the cells were stimulated over a period of 30 min with different combinations of T3 (10-8 M) and/or oPRL (1 µg/ml). No Stat5a DNA binding was seen in the absence of oPRL (with or without T3) (Fig. 4Go, A and B, lanes 1–10). When cells were stimulated with oPRL, a specific association of Stat5a to its DNA binding site was identified (Fig. 4Go, A and B, lanes 11–20). Specificity of Stat5a for its consensus sequence was verified by competition with a 100-fold excess of unlabeled oligonucleotides (Fig. 4Go, A and B, lanes 12 and 17). Adding 1 µg of mStat5a antibodies resulted in the appearance of a supershifted band containing Stat5a bound to DNA (Fig. 4Go, A and B, lanes 13 and 18). With the LHRE probe, no supershifted or shifted material was apparent after addition of human (h)TRß1 antibodies in extracts of control or stimulated cells by T3 (Fig. 4Go, A and B, lanes 4, 5, 9, and 10). This absence of shift seems to be in agreement with a lack of an apparent canonical consensus TRE on LHRE. In nuclear extracts from oPRL and oPRL + T3 treated cells, addition of increasing amounts of hTRß1 antibodies repeatedly diminished the intensity of the shifted band when TRß1 and RXR{gamma} were overexpressed. As illustrated in Fig. 4AGo, quantification of the shift showed a reduction of 12% and 30%, respectively, in oPRL-treated cells (lanes 14 and 15) and in oPRL + T3-treated cells (lanes 19 and 20). HumanTRß1 antibodies did not affect the shifted band when TRß1 and RXR{gamma} were not overexpressed in cells (Fig. 4BGo, lanes 14, 15, 19, and 20). Such reduced binding of shifted materials (Fig. 4AGo) in the presence of TRß1/RXR{gamma} is not fully understood but might represent an unstable low-affinity heterocomplex containing Stat5a, TRß1, and RXR{gamma} associated within the LHRE consensus sequence and/or loosening of specific Stat5a-DNA binding. Similar results were obtained when cells were transfected with Stat5b instead of Stat5a (data not shown). Moreover, addition of TRß1/RXR{gamma} apparently also increased Stat5a DNA binding as shown in our EMSA when nuclear extracts originated from T3 + oPRL- or T3-treated cells, in comparison to cells transfected without TRß1/RXR{gamma} (Fig. 4AGo, lanes 11 and 16; compare with Fig. 4BGo, lanes 11 and 16).



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Figure 4. EMSA Analysis of Stat5a and TRß1 with LHRE Probe or with TRE Probe

A, Cells were transfected with PRLR, Stat5a, and TRß1/RXR and stimulated with oPRL (1 µg/ml) and/or T3 (10-8 M) for 30 min at 37 C. EMSA analysis was performed with 8 µg of nuclear protein incubated with 40,000 cpm of 32P-LHRE-probe. Supershifts were obtained with 1 µg of anti-mStat5a (polyclonal) or 1–4 µg of anti-hTRß1 (monoclonal) antibodies. Competitions were performed with 100-fold excess of unlabeled probe LHRE and films exposed 48 h at -80 C. B, Cells were transfected with only Stat5a and PRLR under the same conditions applied in panel A. Panels A and B were quantified with ImageQuant logiciel, and measurement corresponds to intensity shift percentage relative to lane 16. C, EMSA analysis was performed with 8 µg of nuclear extracts from T3 - and T3 + PRL-treated cells and incubated with 10,000 cpm of the 32P-TREpal probe. Supershifts were estimated after addition of anti-hTRß1 (1 µg) or anti-mStat5a antibodies (1 µg). The competition with cold oligonucleotides was performed as described in panel A.

 
The specificity of supershifted materials by hTRß1 antibodies was confirmed using the same EMSA conditions as described above, with a palindromic (IR0) TRE oligonucleotide probe (5'-AGGCTCAGGTCATGACCTGAGCCC-3') (Fig. 4CGo). TRß1, RXR{gamma}, and Stat5a were overexpressed in 293 cells stimulated during 30 min with T3 and T3 + oPRL. In both groups specific shifts were observed in nuclear cells extracts (Fig. 4CGo, lanes 1–8). In these two treatment conditions the shifted material was supershifted in presence of hTRß1 antibodies (Fig. 4CGo, lanes 4 and 8). A marked inhibition of the shift and supershift was apparent in nuclear extracts from oPRL + T3 (Fig. 4CGo, lanes 5–8) compared with T3-treated cells (lanes 1–4). When mStat5a antibodies were used, no supershifted materials could be seen regardless of the hormonal status of nuclear extracts used (Fig. 4CGo, lanes 3 and 7). For all combinations of treatment, the specificity of the shifted bands was validated by addition of 100-fold excess of unlabeled TRE (Fig. 4CGo, lanes 2 and 6).

Effect of T3 and oPRL on Stat5a and TRß1 in Whole Cell Extracts
Using Western blotting analysis we next investigated the interactions between Stat5a and TRß1 and their phosphorylation status. In all the transfection studies shown in Fig. 5Go, A and B, cells were cotransfected with PRLR, Stat5a, RXR{gamma}, and TRß1. As shown in Fig. 5AGo, the effects of oPRL and T3 on Stat5a phosphorylation was estimated after immunoprecipitation with anti-mStat5a or anti-hTRß1 antibodies and immunoblotted with antiphosphotyrosine antibodies. Tyrosine phosphorylation of Stat5a was only apparent in cells stimulated by oPRL (1 µg/ml) (Fig. 5AGo, Stat5a arrowhead). A lower level of activated Stat5a tyrosine phosphorylation was shown in the cytosol of cells incubated simultaneously with both oPRL and T3 (Fig. 5AGo, Stat5a arrowhead; compare lanes 5 and 7). When the same extracts were immunoprecipitated by anti-hTRß1 antibodies and immunoblotted with antiphosphotyrosine antibodies, phosphorylated TRß1 was apparent in all extracts, regardless of treatment (Fig. 5AGo, TRß1 arrowhead). To further evaluate whether or not coexpressed TRß1 might complex with Stat5a, the ability of TRß1 to coimmunoprecipitate with Stat5a was tested in whole cell extracts after addition of monoclonal hTRß1 antibodies and immunoblotting with polyclonal mStat5a antibodies (Fig. 5BGo). Comparisons were performed using the same extract immunoprecipitated and probed with mStat5a antibodies. Western blot analysis after single immunoprecipitation and immunoblotting with mStat5a antibodies revealed one specific band of 92 kDa. In whole cell extracts immunoprecipitated by hTRß1 antibodies and probed with mStat5a antibodies, Stat5a occurred only in cells stimulated by oPRL or oPRL + T3 (Fig. 5BGo, Stat5a arrowhead). No apparent association of TRß1 with Stat5a could be identified in unstimulated cells (control) or in cells incubated with T3 alone. This TRß1-Stat5a association was also observed in cytoplasmic extracts but was undetectable in the nucleus (data not shown). Expression of endogenous Stat5a and Stat5b in untransfected 293 cells was estimated in whole cell extracts immunoprecipitated and probed with mStat5a or mStat5b antibodies. As shown in Fig. 5CGo, we were not able to detect Stat5b, whereas low levels of Stat5a are present.



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Figure 5. Effects of oPRL and T3 on Stat5a Phosphorylation and Association with TRß1 in Whole Cell Extracts

Immunoprecipitation was performed with 20 µl of protein-A Agarose and polyclonal anti-mStat5a antibodies or 20 µl of protein G-plus Agarose with monoclonal anti-hTRß1 antibodies. The whole cell extracts were imunoprecipitated with anti-mStat5a or with anti-hTRß1 during 2 h and run on a 7.5% SDS-polyacrylamide gel. A, PVDF membranes were incubated with monoclonal antiphosphotyrosine antibodies (Stat5a arrowhead) or monoclonal anti-hTRß1 antibodies (TRß1 arrowhead). B, The same membrane was stripped and immunoblotted with anti-mStat5a antibodies (Stat5a arrowhead). C, Whole cell extracts from untransfected cells were immunoprecipitated with a polyclonal anti-mStat5a or anti-mStat5b and immunoblotted with the same antibody (Stat5a arrowhead) as described above.

 
Effect of TRß1/RXR{gamma} on Stat5a Nuclear Translocation
We further evaluated whether or not TRß1/RXR{gamma} affects the subcellular distribution and nuclear translocation of Stat5a. All transient transfections performed with PRLR and Stat5a were complemented with TRß1 and RXR{gamma} (+TR) or control plasmid pcDNA3 (-TR). Western blots were performed on cytosolic (Fig. 6AGo) and nuclear extracts (Fig. 6BGo). Extracts were immunoprecipitated and immunoblotted with anti-mStat5a antibodies. In the presence of TRß1/RXR{gamma}, decreased Stat5a levels were apparent in all cytosolic extracts compared with controls (Fig. 6AGo). In the nucleus, overexpression of TRß1/RXR{gamma} induced an increased nuclear localization of Stat5a (Fig. 6BGo). In each group of transfected cells (+TR or -TR), hormonal treatments did not apparently modify Stat5a expression in cytosolic (Fig. 6AGo) or nuclear extracts (Fig. 6BGo).



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Figure 6. Overexpression of TRß1/RXR{gamma} Heterodimer Induces Translocation of Stat5a in the Nucleus

A, Cells were transfected with PRLR and Stat5a and complemented with pcDNA3 in groups (-TR, lanes 1, 3, 5, and 7) or complemented with TRß1/RXR{gamma} in groups (+TR, lanes 2, 4, 6, and 8) or with GFP-TR2A in groups (+TR, lanes 1–4, panels C and D). Cells were stimulated as previously described in Fig. 4Go. Immunoprecipitations were performed with a polyclonal anti-mStat5a antibodies on cytoplasmic extracts (A and C, upper panel) or nuclear extracts (B and D, lower panel) (as described in Fig. 5Go). After transfer the blots were probed with an anti-mStat5a (A, B, C, and D, Stat5a arrowhead) or an antiphosphotyrosine (A and B, pTyr arrowhead).

 
The same blots were then probed with antiphosphotyrosine antibodies (Fig. 6Go, A and B). Phosphorylated Stat5a was observed only in cytosolic and nuclear extracts from cells stimulated with oPRL or with oPRL + T3 (Fig. 6AGo, lanes 5–8). In oPRL-treated cells overexpressing TRß1/RXR{gamma}, a decreased amount of phosphorylated Stat5a was apparent in the cytoplasm compared with cells without TRß1/RXR{gamma} (Fig. 6AGo, lane 6 vs. lane 5, pTyr arrowhead). We found an opposite effect in the nucleus (Fig. 6BGo, lane 6 vs. lane 5, pTyr arrowhead). This trend toward Stat5a diminution in the cytoplasm was further lowered in the presence of T3 + oPRL in both groups of transfected cells (Fig. 6Go, A and B, lane 8 vs. 7, pTyr arrowhead). In contrast, increased amounts of phosphorylated Stat5a and Stat5a protein could be found in the corresponding nuclear compartment, when cells were transfected with TRß1/RXR{gamma} and treated by oPRL or T3 + oPRL (Fig. 6BGo, lanes 6 and 8, pTyr arrowhead) than in cells without TRß1/RXR{gamma} (Fig. 6BGo, lanes 5 and 7, pTyr arrowhead) or cells unstimulated by oPRL (lanes 1–4).

In order to ascertain how the NLS-mutated GFP-TR2A/RXR{gamma} partly abrogated the T3-inhibitory effect on PRL-induced LHRE-TK-luciferase expression, we evaluated its role on the subcellular distribution of Stat5a (Fig. 6Go, C and D). Cytoplasmic and nuclear extracts were immunoprecipitated and immunoblotted with anti-mStat5a antibodies. No modification in the amount of Stat5a in the cytosol was evidenced when GFP-TR2A was overexpressed between the different types of treatment (Fig. 6CGo). Overexpression of GFP-TR2A/RXR{gamma} did not induce nuclear localization of Stat5a in control or T3-treated cells (Fig. 6CGo). Thus, overexpression of GFP-TR2A did not apparently affect the amount of Stat5a present in cytosolic or nuclear extracts.

The Effects of Histone Acetylation on T3-Mediated Transcriptional Repression
To assess whether or not histone deacetylase activity is involved in the T3-mediated repression of Stat5a-dependent transcription, we tested the ability of TSA, a potent inhibitor of histone deacetylase, to affect Stat5a-induced promoter activity. Hormonal treatments were simultaneously performed in 293 cells with or without TSA. As shown in Fig. 7Go, in the absence of TSA, T3 addition inhibited PRL-induced LHRE activity by up to 40%. Addition of TSA (1000 nM) potentiated oPRL-induced LHRE promoter activity as illustrated by the 10-fold increase in luciferase expression (Fig. 7AGo). At the dose of TSA used in our study, TSA had no significant effect on promoter activity in the absence of oPRL. In contrast, a clear dose-response effect on oPRL-induced LHRE-TK-luciferase expression was obtained with increased amounts of TSA (50 to 1000 nM) (data not shown). As shown in Fig. 7BGo, T3-dependent repression was almost fully abrogated in the presence of TSA. Thus, our results suggest that recruitment of histone deacetylase activity by liganded TRß1 might be part of its inhibitory effect on LHRE promoter activity.



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Figure 7. Deacetylase Inhibitor TSA Reverses the T3 Inhibitory Effect of TRß1/RXR{gamma} on LHRE-TK-Luciferase Activity

A, Cells were transfected with PRLR, RXR{gamma}, and TRß1. As indicated, cells were stimulated by oPRL (1 µg/ml) and/or T3 (10-8 M) and 1000 nM of TSA added during 10–12 h in the starvation medium (at 37 C). The relative light units were measured and normalized for ß-galactosidase activity. Results are the mean ± SE of three independent experiments, each performed in duplicate. B, Inhibition percentage in oPRL-treated cells was estimated as % of T3-induced reduction of luciferase activity in presence or absence of TSA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present study demonstrates novel functional interactions between TRs and PRL-induced Stat5a transcriptional activity. In the presence of its receptor TRß1 alone, and further with the TRß1/RXR{gamma} heterodimer, T3 induces a clear inhibition of PRL-induced transcription. This T3-dependent TRß1 inhibition was apparent with both Stat5a and Stat5b, using PRLR and GHR on a construct containing six copies of LHRE. These observations could partially explain the observation that high levels of TH have been shown to inhibit PRL-induced mammary gland development and milk production in mammals (6). In recent years, a number of nuclear steroid receptors have been identified that are critical for the transcriptional responses transduced by Stat5. It is well established that phosphorylation of Stat5 plays a central role in stabilizing the processes of dimerization and nuclear translocation upon activation of PRL-induced gene expression (37). However, the mechanisms underlying the formation of an active Stat dimer and its translocation to the nucleus have not been fully elucidated. No NLSs have been yet identified on Stat molecules, raising the question of how they are imported to the nucleus (38).

In our model, after immunoprecipitation and Western blotting with Stat5a antibodies, we showed that TRß1/RXR{gamma}, respectively, decreased Stat5a protein level in the cytosol and increased Stat5a nuclear protein localization, suggesting their association during the process of induced nuclear translocation. This process occurred even in control cells without oPRL and T3 and in T3-treated cells. However, we were not able to show in either of these groups the occurrence of stable association between Stat5a and TRß1 or Stat5 DNA binding on the LHRE. Thus our data suggest that overexpression of TRß1/RXR{gamma} induced protein-protein interaction with Stat5a and increased nuclear translocation and sequestration of a subpopulation of nonactivated Stat5 monomer before any treatments in our model, similar to the model of GR/Stat5 interaction (27). Indeed, similar LHRE- driven luciferase activity was seen in the presence or absence of TRß1/RXR{gamma} in control or oPRL-treated cells (our unpublished observations). Moreover, expression of RXR{gamma} in the absence of TRß1 did not induce inhibition of Stat5a transcriptional activity or changes in nuclear translocation (data not shown). However, RXR{gamma} potentiated TRß1 interaction with translocation of inactivated Stat5a into the nucleus.

The nuclear localization of TRß1 in the absence of ligand remains controversial (36). Recently, an equal distribution of TRß1 isoforms between the nucleus and cytoplasm has been observed in the absence of ligand (36, 39). Alanine substitution of Lys184-Arg185 in the D-domain of the GFP-TR2A mutant, between the DNA and hormone binding domain of TRß1, was shown to inhibit both its nuclear translocation and T3-mediated transactivation (36). Using this mutated construct of TRß1 in our study, a marked attenuation of the T3-inhibitory effect on Stat5a transduction pathways was observed. This loss of T3 inhibition was associated in our Western blotting studies with a lowered amount of Stat5a translocated or sequestrated in the nucleus. These data suggest that nuclear translocation and/or sequestration of latent Stat5a monomers, regardless of the hormonal treatment, might be closely linked to unliganded TRß1 subcellular distribution in our study.

Stat5a phosphorylation plays a critical role in Stat5a dimerization, nuclear translocation, and DNA binding (3). We show that Stat5a and TRß1 could be coimmunoprecipitated in whole cell extracts only when Stat5a phosphorylation was induced by oPRL treatment. Furthermore, expression of TRß1/RXR{gamma} in oPRL-treated cells was associated with increased amount of phosphorylated Stat5a in the nuclear compartment as evidenced in our Western blotting studies. Thus, oPRL-induced Stat5a phosphorylation and dimerization in our study likely strengthened the binding affinity involved in the physical interactions between Stat5a and TRß1 molecules.

Several functional interactions involving Stat5a or Stat5b and nuclear receptors from the steroid/thyroid receptor family members have been published (25, 26, 27, 28, 29, 30). Among them, reports on GR and PR suggested that those nuclear receptors and Stat might converge and physically interact in the cytoplasm, thereby enhancing Stat5a entry into the nucleus (27, 28). Moreover, overexpression of GR enhanced Stat5 tyrosine phosphorylation and DNA binding (27), an effect possibly related to the predominantly cytoplasmic subcellular localization of GR (40). Interestingly, when overexpressing the GR, Cella et al. (30) found 1.5 times more GR or Stat5 present in the nucleus, respectively, in cells stimulated by oPRL or dexamethasone compared with unstimulated cells, suggesting that inactive partner recruitment occurred before nuclear translocation. Similar results were obtained with the PR (28). Progesterone alone in the T47Dco breast cancer cell line also induced nuclear translocation of Stat5, suggesting that liganded PR, like GR, may be implicated as cotransporter of Stat5 in the nucleus.

Although increased phosphorylated Stat5a was present in the nucleus of oPRL cells transfected with TRß1/RXR{gamma}, we were unable to detect, with EMSA analysis and specific antibodies against TRß1, any supershifted molecular complex representing a stable association of Stat5a with TRß1. Nevertheless, addition of increased amounts of TRß1 antibodies disrupted the shifted DNA binding of Stat5a from oPRL and T3 + oPRL nuclear extracts when TRß1/RXR{gamma} were overexpressed but not in their absence. This latter observation clearly supports the recruitment of TRß1 by activated Stat5a dimer within its cognate DNA binding site. However, our data differ somewhat from those obtained on the cross-talk between ER and Stat5. Overexpression of ER in the presence of oPRL and estrogen was associated with a clear inhibition of Stat5-DNA binding (27). In these studies, the inhibitory effect of ER on Stat5 DNA binding was shown to arise from a decreased Stat5a phosphorylation. Our data also differ from the interactions between Stat5a and b with GR, which are among the best studied compared with other members of this nuclear receptor superfamily (26, 27, 30, 41, 42). GR does not bind to, but directly associates with, Stat5 on its DNA response element (LHRE) in the promoter of the ß-casein gene. Treatment with oPRL and dexamethasone induced the formation of a GR/Stat5/DNA complex as confirmed in EMSA where supershifted materials were seen using specific antiserum against either Stat5 or the GR (26). Thus, the hypothesis we put forward to explain such discrepancies in the literature is that the complex containing Stat5a and TRß1 or Stat5 and ER on a Stat5 responsive element (LHRE) possibly interacts with a lower or a weaker DNA binding affinity compared with the heterocomplex containing GR and Stat5a. It is important to mention that among members of the nuclear steroid/thyroid receptor family, GR and PR differ from thyroid and ERs in their recognition of DNA response elements (24, 43, 44). Several half-GREs have been mapped in the ß-casein promoter (45), but no half-site TRE that binds TR and ER (AGGTCA) has been reported. Interestingly, PRs and GRs potentiated (25), whereas ER repressed (25), Stat5 transcriptional activity as TRß1. However, we do not fully understand how T3 or estrogen negatively coordinates Stat5 DNA binding and gene transcription through its responsive element. However, the data presented here are consistent with the hypothesis that TRß1/RXR{gamma} and T3 in the presence of PRL enhance dimerization of Stat5a in a functional antagonistic heterocomplex with TRß1, thus increasing nuclear translocation and DNA binding activity of this complex at the LHRE site present in the promoter of the ß-casein gene. Such increased DNA-binding activity of Stat5a dimer, as a result of increased Stat5a/TRß1/RXR{gamma} nuclear translocation and/or its decreased nuclear turnover, might represent one of the functional consequences in TRß1/RXR{gamma}-mediated inhibition at the promoter level. A similar model was demonstrated with GR and Stat5, PRL-induced Stat5 dimerization resulting in an increased nuclear localization of unliganded cytoplasmic GR (27). Furthermore, physical interactions between TRß1 and Stat5a might be only one component, the transcriptional antagonism exhibited by this complex likely involving recruitment of other partners to be operable on PRL-dependent target genes. The experiments presented here strongly suggest the occurrence of protein/protein interactions between Stat5a and TRß1 and the transcriptional machinery in oPRL- and oPRL + T3 -stimulated cells. Consequently, the carboxy-terminal ligand binding domain of TRß1 was likely not involved in this association, since a clear inhibition of oPRL transcriptional activity could be obtained only upon addition of T3.

In our EMSA analysis using an idealized TREpal we repeatedly observed DNA binding of TRß1 supershifted by hTRß1 antibodies, in nuclear extracts from T3- and T3 + oPRL-treated cells. Additionally, a clear inhibition of the shifted and supershifted TRE probe was seen in oPRL and oPRL + T3 nuclear extracts compared with those originating from T3-treated cells. This observation seems relevant since GH significantly inhibited a T3-induced TRE-CAT transcriptional activity when TRß1 was overexpressed (46). Thus, it appears likely that the physical cross-talk of TRß1 and Stat5a in our studies also affects thyroid hormone transduction pathway. Such cross-talk observed only during hormonal treatment might be stabilized by multiprotein complex components in the transcriptional machinery such as nuclear corepressor (NcoR) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) with associated HDACs or sin3 and Rpd3 histone deacetylase activity or nuclear coactivator (NCoA) with histone acetyltransferase activity (47). Members of the Stats family have been shown to interact directly with the related coactivator p300 or with N-myc interactor (Nmi), a new auxiliary protein downstream of CREB binding protein (CBP)/p300 (48). CBP/p300 or the steroid receptor coactivator SRC-1 does not appear to participate in the Stat5a-mediated suppression of the inhibitory effect of Stat5a on glucocorticoid or PPAR{alpha} responsive elements (41, 46). However, we found in our present study that TSA, a potent deacetylase inhibitor, potentiated PRL-induced gene transcription and abrogated T3 inhibition on Stat5a transcriptional activity. TSA markedly stimulated PRL transduction pathway, suggesting that interplay between NCoA and NCoR both occurred in Stat5a transactivation. TRH and TSH both have a negative responsive element (nTRE) allowing negative feedback regulation by TRß1 in the presence of T3 (19, 20, 49). Characterization of nTRE consensus nucleotide sequences differs in TSH and TRH promoter (49, 50). Depending on the nTRE present at the promoter level, unliganded or liganded TRß1/RXR{gamma} exerted its negative regulation by recruitment of histone deacetylase. Thus, in our model, liganded TRß1 could allow the recruitment of a complex that contains histone deacetylase activity in its inhibitory effects on Stat5a transcription activity as already postulated in the negative feedback regulation of TSH (49).

Finally, the present data support the concept that those nuclear receptors affecting Stat5 signaling have two key characteristics: they all contain putative NLSs and all interact with the transcriptional machinery either directly or indirectly (38). We postulate that the interactions between Stat5a and TRß1 described in the present study may be part of the mechanisms implicated in the TH-induced inhibition of mammary gland development. Similar antagonistic cross-talk, but on a T3 responsive element, might be also part of the antagonistic effect elicited by PRL during TH-induced metamorphosis in amphibians (13).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
As previously described (51), the LHRE-TK-luciferase construct is a fusion gene carrying six copies of the LHRE DNA element present on the ß-casein gene promoter upstream of the TK minimal promoter and the coding region of the luciferase reporter gene. The wild-type ß-casein-luciferase plasmid was a gift from Dr. Goffin (Paris, France). The construct contains a fragment of the proximal promoter of rat ß-casein extending from -2300/+490 inserted into NheI site of luciferase reporter plasmid pGL2-E (51). Cytomegalovirus-based expression plasmids were used for the pR/CMV vector (Invitrogen, San Diego, CA) containing cDNAs encoding the long form of the rat PRLR (35) and the pcDNA3-monkey (mk)-GHR (53). Expression plasmid DNA, pXM-MGF/Stat5a, encoding bovine MGF/Stat5a, was obtained from Dr. B. Groner (Freiburg, Germany). GFP-Stat5b expression plasmid was introduced in a PEGFP-CI vector with a cytomegalovirus (CMV) promoter kindly provided by Dr. J. Herrington (University of Michigan, Ann Arbor, MI) (54). The human plasmid pSG5-TRß1 was provided by Pr D. Stehelin (Lille, France). It was generated by subcloning the 1.7-kb EcoRI fragment of TRß1 which contains the cDNA for human erb-A into the pSG5 (55). The expression vector pCMX-RXR{gamma} was provided by D. Mangelsdorf (Dallas, TX). PCI-nGFP-TRß1 and the NLS-mutated construct pCI-nGFP-TR2A were provided by Dr. S.-Y. Cheng (36) (NIH, Baltimore, MD). CMV ß-galactosidase cDNA was used as internal control to normalize LHRE-luciferase and ß-casein-luciferase expression in cell-transient transfection assays (52), and pcDNA3 vector (Promega Corp., Madison, WI) was used for DNA complementation. Ovine PRL (NIDDK-oPRL-20) was a gift from the National Hormone and Pituitary/NIDDK program (Baltimore, MD), T3 was purchased from Sigma (St. Louis, MO), and TSA was purchased from Wako Pure Chemical Industries Ltd. (Richmond, VA). Monoclonal anti-hTRß1 (sc-737 or sc-737X for EMSA), polyclonal anti-mStat5a (sc-1081 or sc-1081X for EMSA), and polyclonal anti-mStat5b (sc-835-G or sc-835X for EMSA) used for Western blot were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). We used also a monoclonal antiphosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY). Protein levels were determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc. Hercules, CA) (56).

Cell Culture and Transfections
The human embryonic kidney fibroblast HEK 293 cell line was used for transient transfection and maintained in DMEMnut F12 complemented with 10% FCS, 2 mM glutamine, and antibiotics. Cells were split into six-well plates at a density of 800.000 cells per well (~70% confluency) in DMEM (4.5 g/liter of glucose, 10% FCS, 2 mM glutamine, antibiotics) before being transiently transfected using the calcium phosphate technique (57) with 0.1 µg/well of LHRE-TK-luciferase and ß-galactosidase, 0.05 µg/well of PRLR, TRß1 (GFP-TRß1 or GFP-TR2A), and RXR{gamma}. Within 15 h after transfection, the cells were starved in the absence of FCS in DMEM (4.5 g/liter of glucose) for an additional 7 h of starvation. Cells were then stimulated during 10–12 h for functional test by oPRL (1 µg/ml), T3 (10-8 M), or TSA (1000 nM) added alone or in combination to culture medium. Luciferase activity was measured in relative light units (RLU) and normalized by the estimated ß-galactosidase activity, as previously described (52).

Cytosolic and Nuclear Cell Extracts Preparation
HEK293 cells were grown in 100-mm culture dish. Transient transfection was performed as above using 1 µg of cDNA encoding the PRLR, Stat5a, TRß1, and RXR{gamma}. After transfection cells were treated during 30 min with oPRL (1 µg/ml) and with T3 (10-8 M). Cells were subsequently lysed with 1 ml of lysis buffer (10 mM Tris-HCL, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na3VO4, 10% glycerol, 0.5% Triton X-100) containing a cocktail of protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml pepstatin, 2 µg/ml leupeptin, 5 µg/ml aprotinin) for 30 min at 4 C. All extracts were aliquoted and frozen at -80 C.

Cells were washed with 1x PBS to remove media and serum, scraped, and collected in 1 ml of 1x PBS with 1 mM of Na3VO4. Cells were pelleted and resuspended by repeated pipetting in 4 volumes of hypotonic buffer (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) supplemented with 1 mM Na3VO4, 20 mM NaF, and 0.2 mM PMSF. The cytoplasmic supernatant was collected and nuclear pellet was resuspended in 1 volume of hypertonic buffer on ice 10–15 min (20 mM HEPES-KOH at pH 7.9, 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA) and complemented with 0.2 mM PMSF, 0.2 mM phosphatase, 20 mM NaF, and 1 mM Na3VO4. Extracts were centrifuged at 10.000 rpm at 4 C for 10 min to remove nuclear debris. All extracts were aliquoted and frozen at -80 C. Each aliquot was thawed only once.

Immunoprecipitation and Western Blotting
For immunoprecipitation, 500 µl of lysates were incubated with 2 µl of anti-mStat5a antibodies (0.8 µg/ml) and anti-hTRß1 (1.2 µg/ml) and mixed with 30 µl of Protein A-Agarose or Protein G-plus (Santa Cruz Biotechnology, Inc., TEBU) for 2 h with shaking. The immunoprecipitate was collected by centrifugation, washed in lysis buffer, and boiled for 5 min in sample buffer (125 mM Tris, pH 6.8, 5% SDS, 10% ß-mercaptoethanol, 20% glycerol). Immunoprecipitated proteins were separated by SDS-PAGE (7.5%) for 2 h at 30 mA. The gel was transferred onto polyvinylidene difluoride (PVDF) transfer membrane (Polyscreen, NEN Life Science Products, Boston, MA), and probed with anti-mStat5a or anti-mStat5b (Upstate Biotechnology, Inc., 1:1000), anti-hTRß1 (Upstate Biotechnology, Inc., 1:1000), antiphosphotyrosine (Upstate Biotechnology, Inc., 1:10000) conjugated with an antimouse or an antirabbit IgG-horseradish-peroxidase (1:5000) and visualized by the ECL detection system (Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min at room temperature.

Electrophoretic Gel Mobility Shift Assay (EMSA)
The same experimental procedure was used for cell culture as described above. The single-strand sequences of LHRE used in our assay were, respectively: 5'-AGGCTGACTTCTTGGAATTAGCCC-3'(sense), 5'-GGGCTAATTCCAAGAAGTCAGCCT-3'(antisense). These probes were provided by Genaxis (Saint-Cloud, France). The palindromic TREpal was used for TRß1 DNA-binding. This sequence contain a TRE underlined as follow: 5'-AGGCTCAGGTCA TGACCTGAGCCC-3' (sense) and 5'-GGGCTCAGGTCATG ACCTGAGCCT-3' (antisense).

The two complementary strands were annealed and end-labeled with [32P]-dATP{gamma} by the Polynucleotide Kinase T4 and purified by P-6 Micro Bio spin columns (Bio-Rad Laboratories, Inc.). Eight micrograms of nuclear extracts were used per reaction, 2 µg of poly(dI)-poly(dC) (Pharmacia Biotech, Piscataway, NJ) were added to 2 µl of migration buffer (50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol), and total volume was adjusted at 20 µl. The sample were incubated 30 min at 4 C with 40,000 cpm of probe. When competitor was added to specific concentration as indicated, they were included with extracts during the incubation of labeled probe. For all antibodies, 1 µg to 4 µg/reaction were used and incubated 30 min before the reaction binding. Reactions were resolved on 4% polyacrylamide (38:2 acrylamide/bis-acrylamide, Interchim) gels containing 0.25x TBE. Gels were pre-run at 50 V overnight and run 1 to 1.30 h at room temperature. They were dried on wathman and analyzed with Kodak (Eastman Kodak, Rochester, NY) film after 48 h exposure at -80 C. We used the ImageQuant logiciel (Molecular Dynamics, Inc., Sunnyvale, CA) for calculating the percentage of shift intensity.


    ACKNOWLEDGMENTS
 
The authors wish to thank the National Institute of Diabetes, Digestive and Kidney Diseases, NIH (Bethesda, MD), Drs. A. Begue, R. M. Evans, S.-Y. Cheng, C. Carter-Su, and L. Sachs for helpful discussion and for their kind gifts of DNA and reagents.


    FOOTNOTES
 
Address requests for reprints to: Dr. M. Edery, INSERM Unité 344, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris cedex 15-France.

Received for publication August 18, 1999. Revision received May 31, 2000. Accepted for publication June 1, 2000.


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 DISCUSSION
 MATERIALS AND METHODS
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