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Endocrinology Vol. 141, No. 2 606-614
Copyright © 2000 by The Endocrine Society

ARTICLES

Differential Effects of Acute and Chronic Exposure to Interferon-{gamma} on Cyclic Adenosine 3',5'-Monophosphate Response Element-Regulated Gene Expression1

Arturo P. Saavedra, Lisa A. Cass, Gregory V. Prendergast and Judy L. Meinkoth

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Judy L. Meinkoth, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084. E-mail: meinkoth{at}pharm.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TSH stimulates proliferation and maintains differentiated function in thyroid follicular cells. The mitogenic activity and the stimulatory effects of TSH on thyroid-specific gene expression are impaired by interferon-{gamma} (IFN{gamma}); however, the mechanisms for these effects have not been elucidated in detail. We examined the effects of IFN{gamma} on acute responses to TSH in rat thyroid cells. IFN{gamma} did not impair TSH-stimulated p70/p85 ribosomal protein S6 kinase (p70/p85s6k) activity or cAMP response element (CRE)-regulated gene expression, although it inhibited DNA synthesis and thyroglobulin expression, effects measured over a more prolonged time course than those on kinase activity and reporter gene expression. Unexpectedly, when cells were chronically exposed to IFN{gamma}, CRE-lacZ promoter activity was decreased, whereas other cAMP-mediated signals, such as p70/p85s6k activity and CRE-binding protein phosphorylation, were unaffected. Activating protein-1-regulated promoters were also impaired by IFN{gamma} treatment, but with kinetics that differed from those of CRE-regulated promoters. Neither acute nor chronic treatment with interleukin-1ß impaired cAMP signaling, indicating that the effects of IFN{gamma} are specific. These studies identify CRE- and activating protein-1-regulated promoters as targets of IFN{gamma} in thyroid cells and fibroblasts. IFN{gamma}-mediated inhibition of these promoters, in addition to those containing thyroid-specific transcription factor-1-binding sites, may contribute to the profound effects of IFN{gamma} on thyroid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TSH COORDINATELY regulates proliferation and differentiated function in thyroid follicular cells. TSH stimulates the expression of thyroid-specific genes, including the TSH receptor (TSHR) (reviewed in Ref. 1), thyroglobulin (Tg) (2, 3, 4, 5), thyroid peroxidase (5, 6), iodothyronine 5-deiodinase (7), and the sodium-iodide symporter (8, 9). Cytokines, including interferon-{gamma} (IFN{gamma}), interleukin-1ß (IL-1ß), and tumor necrosis factor-{alpha}, are believed to participate in the development and progression of thyroid autoimmunity. IFN{gamma} increases the autoantigenicity of thyrocytes by up-regulating MHC class I expression (10) and by inducing aberrant expression of MHC class II antigens (11, 12, 13, 14, 15, 16, 17). Moreover, the expression of several genes under regulatory control by TSH is inhibited by IFN{gamma}. In turn, TSH impairs IFN{gamma}-stimulated Fas expression (18). In addition to opposing effects on gene expression, TSH and IFN{gamma} exert differential effects on proliferation, where IFN{gamma} has been shown to inhibit TSH-stimulated proliferation (12, 19, 20).

Thyroid-specific transcription factor-1 (TITF-1; previously termed TTF-1) (21) has been identified as one molecular target of IFN{gamma} in FRTL-5 cells where IFN{gamma} decreases TITF-1 DNA-binding activity on the TSHR promoter (22). In human thyroid cells, IFN{gamma} activates a nuclear protein that binds to the Tg promoter and diminishes Tg expression (23). Given the multitude of the effects of IFN{gamma} on thyroid cells, it seems likely that there are additional mechanisms through which IFN{gamma} impairs gene expression and proliferation. Gene expression from a mutant TSHR promoter deleted of the TITF-1-binding site was impaired by IFN{gamma}, suggesting that there are additional targets of IFN{gamma}-mediated repression. In support of this view, we demonstrate for the first time that cAMP response element (CRE) promoter activity is a target of IFN{gamma}-mediated inhibition, but only when thyroid cells are chronically treated with this cytokine. Interestingly, other protein kinase A (PKA)-dependent nuclear effects stimulated by TSH, including p85s6k activation and CRE-binding protein (CREB) phosphorylation, were not impaired by chronic exposure to IFN{gamma}. Activating protein-1 (AP-1)-regulated gene expression was also inhibited by chronic treatment with IFN{gamma}. Unlike CRE-regulated gene expression, however, AP-1 promoter activity was modestly, but reproducibly, reduced by acute treatment with IFN{gamma}. These results demonstrate that the effects of IFN{gamma} are not limited to TITF-1-regulated promoters, and that there are multiple, temporally distinguishable mechanisms through which IFN{gamma} affects thyroid cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat recombinant (r) IFN{gamma} was obtained from Genzyme Corp. (Cambridge, MA), and rat rIL-1ß was purchased from Sigma (St. Louis, MO). Cell culture reagents, crude bovine TSH, forskolin, and 8-bromo-cAMP (8BrcAMP) were obtained from Sigma. FCS and calf serum were purchased from Life Technologies, Inc. (Gaithersburg, MD), and BSA was obtained from Bayer Corp. (Kankakee, IL).

Cell culture
Wistar rat thyroid (WRT) cells were maintained in Coon’s modified Ham’s F-12 medium supplemented with crude bovine TSH (1 mU/ml), insulin (10 µg/ml), transferrin (5 µg/ml), 5% calf serum, and antibiotics (3H medium). Cells expressing cAMP response element (CRE)- and AP-1-regulated lacZ genes (WRT CRE or WRT AP-1, respectively) (24, 25) were maintained in 3H containing G418 (150 µg/ml). BALB/c and Rat2 fibroblasts expressing similar reporter genes were maintained in DMEM containing 10% FCS and G418 (150 µg/ml). The lacZ gene expression was measured as described previously (24, 25). Rat embryonic fibroblasts (REF52) were maintained in DMEM containing 10% FCS. For DNA synthesis studies, thyroid cells were grown to 80% confluence and then incubated in basal medium (Coon’s modified Ham’s F-12 medium containing antibiotics and 0.2% BSA) further supplemented with insulin (0.5 µg/ml) for 48 h. Under these conditions, WRT cells were quiescent, as assessed by bromodeoxyuridine (BrdU) incorporation and FACS cell cycle analysis. DNA synthesis was assessed by BrdU incorporation as described previously (26). REF52 cells were rendered quiescent by incubation in serum-free DMEM further supplemented with insulin (0.5 µg/ml) for 24 h and then stimulated with 10% FCS in the presence of BrdU for 24–30 h.

FACS analysis
WRT cells incubated in basal medium for 48 h and exponentially growing REF52 cells were treated with IFN{gamma} (500 U/ml) for 48 h. Cells were trypsinized and collected by centrifugation. For MHC class I (RT1A) expression, 1–2 x 105 (WRT) or 3–6 x 104 (REF52) cells were incubated with a monoclonal antibody to nonpolymorphic determinants of rat MHC class I antigen (RT1A; 5–20 µg/ml; PharMingen, San Diego, CA) for 2 h at 4 C and with secondary antibody (7.5 µg/ml) for 1 h at 4 C. Cells were collected, washed three times in PBS, and resuspended in 300 µl PBS before analysis by the Wistar Institute cytometry facility (Philadelphia, PA).

Immunostaining
Cells on coverslips were grown to 80–90% confluence and incubated in basal medium for 6–8 days to abolish Tg expression as we have previously described (26, 27). Cells were stimulated with TSH (1 mU/ml) or 8BrcAMP (1 mM) in the presence or absence of IFN{gamma} (100–500 U/ml) or IL-1ß (200 U/ml) for 48 h. After fixation in MeOH for 2 min at -20 C, cells were incubated with a polyclonal antibody to Tg (1:400; DAKO Corp., Carpinteria, CA) for 1 h at 37 C and then with a biotinylated antirabbit secondary antibody (1:450) and Texas Red-streptavidin (1:200). CREB phosphorylation was assessed by immunostaining with a phospho-specific antibody raised to serine 133 (Upstate Biotechnology, Inc., Lake Placid, NY). Cells were incubated in basal medium for 48 h in the presence or absence of IFN{gamma} (100 U/ml) and then stimulated with TSH (1 mU/ml) or 8BrcAMP (1 mM) for 90 min. Cells were fixed in 3.7% formaldehyde/PBS for 20 min, permeabilized with 0.2% Triton, and stained with the phospho-specific CREB antibody (1:50) followed by FITC-antirabbit IgG (1:200). Cells were examined by fluorescence microscopy, and photomicrographs were exposed for identical times.

Western blot analysis
To determine Tg expression by Western blot analysis, cells were disrupted in 1% SDS, 1 mM Na3VO4, and 10 mM Tris, pH 7.4, at 95 C; sheared by three passages through a 26-gauge needle; and boiled for 5 min. Seventy-five micrograms of cell proteins were resolved in 6.75% polyacrylamide gels and transferred to polyvinylidene difluoride membranes, and membranes were blocked overnight in 5% nonfat dry milk, 0.1% Tween, and PBS. After incubation with the Tg antibody (2 h, 25 C, 1:800) and secondary antibody, Tg expression was detected with CDP star (New England Biolabs, Inc., Beverly, MA). For p70/85s6k mobility assays and S6 phosphorylation, cells were disrupted in lysis buffer (10 mM KPO4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 2 mM dithiothreitol, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM Pefabloc (a serine protease inhibitor, Pentapham AG, Basel, Switzerland), 10 µg/ml aprotinin, and leupeptin at 4 C for 20 min. Soluble proteins were denatured by boiling in Laemmli sample buffer, resolved on 6.75% (p70/p85s6k) or 12.5% SDS-PAGE (S6), and transferred to polyvinylidene difluoride membranes. Membranes were blocked as described above and incubated for 2 h with a polyclonal antibody to p70/85s6k (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or to a phosphorylated S6 peptide (1:5000; provided by Dr. M. Birnbaum). Incubation with secondary antibodies and detection were performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
WRT cells respond to IFN{gamma} with increased MHC I expression
The effects of IFN{gamma} on WRT cells have not been previously reported. To determine whether these cells respond to IFN{gamma}, we first examined its effects on MHC class I gene expression, which is increased by IFN{gamma} in FRTL-5 cells (10). Using an antibody to nonpolymorphic determinants of rat MHC class I antigens (RT1A), WRT cells were found to express RT1A on the cell surface by FACS analysis (Fig. 1Go). Treatment with IFN{gamma} for 48 h dramatically up-regulated RT1A expression in WRT cells in basal medium (mean fluorescence intensity, 36.1) compared with that in vehicle-stimulated cells (mean fluorescence intensity, 2.4). These results demonstrate that, similar to its effects in FRTL-5 cells (10) and in human thyrocytes (5, 15), WRT cells respond to IFN{gamma} with increased MHC antigen expression.



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  		Figure 1. IFN{gamma} induces MHC class I (RT1A) expression in WRT and REF52 cells. Treatment with IFN{gamma} (500 U/ml), but not diluent, for 48 h dramatically increased RT1A expression in WRT cells in basal medium and in exponentially growing REF52 cells. Three experiments were performed with similar results. IFN{gamma} exerted similar effects in REF52 cells arrested for 24 h in the absence of serum (data not shown).

 
IFN{gamma} represses cAMP-induced Tg expression and DNA synthesis
IFN{gamma} suppresses Tg expression in FRTL-5 cells (28, 29) and human thyrocytes (30), where IFN{gamma} also impairs Tg secretion (5, 31). To determine whether IFN{gamma} altered Tg expression in WRT cells, immunostaining and Western blotting experiments were performed. Tg expression was very low in cells maintained in basal medium for 6–8 days (Fig. 2AGo, panel A), as we have previously reported (26, 27). When added to cells starved in this way, IFN{gamma} did not significantly increase Tg expression (Fig. 2AGo, panel B). Serum also failed to stimulate Tg expression (data not shown). When added together, however, IFN{gamma} and serum collaboratively increased Tg expression (Fig. 2AGo, panel C). TSH (Fig. 2AGo, panel D) and 8BrcAMP (Fig. 2AGo, panel G) markedly stimulated Tg expression. Unlike the effects observed with serum, inclusion of IFN{gamma} together with TSH (Fig. 2AGo, panel E) or 8BrcAMP (Fig. 2AGo, panel H) reproducibly abolished Tg expression. These effects were specific, in that treatment with IL-1ß had no effect on cAMP-mediated Tg expression (Fig. 2AGo, F and I). These results demonstrate that IFN{gamma} elicits opposing effects on Tg expression stimulated by TSH and 8BrcAMP (where it decreases Tg expression) and serum (where it enhances Tg expression). In studies conducted in complex medium that contains TSH and serum, effects of IFN{gamma} on Tg expression may reflect the summation of its effects on individual signaling pathways activated by each component in the medium.



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  		Figure 2. IFN{gamma} represses cAMP-stimulated Tg expression. A, Photomicrographs of representative fields of WRT cells maintained in basal medium for 8 days (A) and cells subsequently treated with IFN{gamma} (B), IFN{gamma} and 5% calf serum (C), TSH (D), IFN{gamma} and TSH (E), IL-1ß and TSH (F), 8BrcAMP (G), IFN{gamma} and 8BrcAMP (H), and IL-1ß and 8BrcAMP (I). The following concentrations were used: IFN{gamma}, 500 U/ml; IL-1ß, 200 U/ml; TSH, 1 mU/ml; and 8BrcAMP, 1 mM. All treatments were given for 48 h. The results shown are from a representative experiment of five that yielded similar findings. Photomicrographs were exposed for identical times, allowing direct comparison of the number of cells expressing Tg under various treatments. Basal levels of Tg expression were repressed by serum, and no immunopositive cells were seen under these conditions. Similar effects of serum on Tg expression were previously reported in calf thyrocytes (reviewed in Ref. 46). B), Lysates (75 µg) prepared from cells incubated in basal medium for 8 days and treated for 48 h as indicated were subjected to Western blotting with a Tg-specific antibody. Concentrations were the same as those used above. Densitometric analysis performed using a ScanMaker4 (Microtek) and NIH Image software (version 1.6) revealed that TSH and 8BrcAMP increased Tg expression by 3.1- and 2.8-fold, respectively. Inclusion of IFN{gamma} reduced TSH-stimulated Tg expression to 2-fold over basal levels, whereas IL-1ß had no effect (3.2-fold). 8BrcAMP-stimulated Tg expression was reduced to background levels (0.5-fold basal levels) in the presence of IFN{gamma}. Two experiments were performed with similar results.

 
To corroborate the immunostaining studies, the effects of IFN{gamma} on Tg expression were examined in Western blotting experiments. Tg expression was extremely low in cells incubated in basal medium for 6–8 days (Fig. 2BGo). TSH and 8BrcAMP each stimulated a 3-fold increase in Tg expression. In the presence of IFN{gamma}, TSH-stimulated Tg protein levels were decreased to 2-fold of basal levels, whereas Tg expression stimulated by 8BrcAMP was reduced to background levels. The inhibitory effects of IFN{gamma} were specific, as treatment with IL-1ß did not affect TSH- or 8BrcAMP-stimulated (data not shown) Tg expression.

To investigate the effects of IFN{gamma} on cell proliferation, DNA synthesis studies were performed. IFN{gamma} markedly impaired DNA synthesis in response to TSH, 8BrcAMP, and serum (Fig. 3AGo). In contrast, IL-1ß did not inhibit DNA synthesis (serum data not shown). The inhibitory effects of IFN{gamma} were not due to nonspecific effects on BrdU incorporation, because serum-stimulated DNA synthesis in REF52 cells, which respond to IFN{gamma} with increased MHC I expression (Fig. 1Go), was not inhibited by IFN{gamma} (Fig. 3BGo). These findings in WRT cells agree with those reported in FRTL-5 (19, 20) and related rat cells (12) as well as in human fetal thyroid cells (31).



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  		Figure 3. IFN{gamma} inhibits cAMP- and serum-stimulated DNA synthesis. A, Quiescent WRT cells were stimulated with TSH (1 mU/ml), 8BrcAMP (1 mM), and FCS (10%) in the presence and absence of IFN{gamma} (500 U/ml) or IL-1ß (200 U/ml) for 48 h. B, REF52 cells were stimulated with FCS (10%) in the presence and absence of IFN{gamma} (500 U/ml) for 30 h. More than 200 cells were scored for each condition. The results shown are the mean ± SE of 3–5 experiments performed with similar results. Error bars are not shown for SE less than 0.045.

 
IFN{gamma} effects on CRE-regulated gene expression
IFN{gamma} has been reported to impair AP-1 promoter activity through competition between STAT1{alpha} (signal transducer and activator of transcription-1{alpha}) and AP-1/ets transcription factors for limiting amounts of CREB-binding protein (CBP) and p300 (32). Because CRE-regulated gene expression is CBP dependent, we examined whether acute treatment with IFN{gamma} could impair CRE promoter activity. WRT cells expressing an integrated CRE-regulated lacZ gene were used for these experiments. These cells have been extensively characterized (25, 26, 27, 33) and respond exclusively and specifically to cAMP-elevating agents. As expected, CRE-regulated gene expression was undetectable in cells maintained in basal medium for 24 h (Fig. 4AGo, panel A) and was induced to high levels after treatment with TSH for 6 h (Fig. 4AGo, panel B). Inclusion of IFN{gamma} (Fig. 4AGo, panel C) or IL-1ß (Fig. 4AGo, panel E) had no effect on CRE-regulated gene expression when added acutely, i.e. together with TSH for 6 h. Even when added 60 min before the cAMP-elevating agents, IFN{gamma} did not impair CRE-regulated gene expression (data not shown). Unexpectedly, when WRT CRE cells were pretreated with IFN{gamma} for 48 h before stimulation with TSH (chronic treatment; Fig. 4AGo, panel D), CRE-regulated gene expression was markedly impaired. In contrast, chronic exposure to IL-1ß (Fig. 4AGo, panel F) had no effect on TSH-stimulated gene expression. Similar results were obtained with 8BrcAMP and cholera toxin (data not shown). These results are summarized in Fig. 4BGo.



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  		Figure 4. Chronic, but not acute, IFN{gamma} treatment represses CRE-regulated gene expression. A, Photomicrographs of representative fields of WRT CRE cells incubated in basal medium for 24 h (A) and cells treated with TSH (1 mU/ml; B), TSH and IFN{gamma} (500 U/ml; C) added concomitantly (acute treatment), and IFN{gamma} for 48 h (chronic treatment; D) followed by TSH for 6 h. Treatment with IL-1ß (200 U/ml; E) together with TSH or 48 h before TSH (F) were included as controls. Three-five experiments were performed with similar results. B, The mean ± SE percentage of cells expressing ß-galactosidase under each condition were determined. Results were obtained from three to five experiments that yielded similar findings.

 
To determine the shortest time of IFN{gamma} treatment sufficient to impair CRE-regulated transcription, time-course experiments were performed. Pretreatment with IFN{gamma} (100–500 U/ml) for 1, 2, or 6 h had no effect on CRE-regulated gene expression. Treatment for 16, 24, or 48 h impaired gene expression to similar levels (data not shown). The inhibitory effects of chronic exposure to IFN{gamma} were not restricted to thyroid cells. Chronic, but not acute, treatment with IFN{gamma} reduced CRE-regulated lacZ gene expression in BALB/c and Rat2 fibroblasts stimulated by forskolin and IBMX (data not shown). These results indicate that CRE-regulated promoters are targets of chronic, but not acute, IFN{gamma} effects.

Acute treatment with IFN{gamma} impairs AP-1-regulated gene expression
To determine whether the chronic effects of IFN{gamma} were restricted to CRE-regulated promoters, effects on AP-1-regulated gene expression were examined. WRT cells expressing an AP-1-regulated lacZ gene (WRT AP-1) were incubated in basal medium in the presence and absence of IFN{gamma} for 24 h and subsequently treated with phorbol ester (TPA) for 6 h. Chronic IFN{gamma} treatment dramatically impaired AP-1-regulated gene expression (Fig. 5AGo, panels G and H). Unlike its effects on CRE-regulated genes, however, acute treatment with IFN{gamma} modestly, but reproducibly, decreased AP-1 promoter activity (Fig. 5AGo, panels E and F), results consistent with an earlier report in macrophages (32). Consistently, acute and chronic IFN{gamma} treatment impaired AP-1 promoter activity in Rat2 fibroblasts (Fig. 5BGo, panels B and C). These results indicate that IFN{gamma} impairs transcriptional activation through mechanisms that are temporally separable: acute effects on AP-1-regulated promoters and chronic effects on CRE-regulated promoters.



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  		Figure 5. IFN{gamma} represses AP-1-regulated gene expression. A, Photomicrographs of two independent experiments, performed in duplicate, of WRT AP-1 cells incubated in basal medium for 24 h (A and B) and cells treated with TPA (100 ng/ml) for 6 h in the absence (C and D) or presence (E and F) of IFN{gamma} treatment for 60 min before stimulation with TPA (acute treatment) or IFN{gamma} treatment for 24 h before treatment with TPA (chronic treatment; G and H). Three experiments were performed with similar results. B, Rat2 fibroblasts expressing an integrated AP-1 lacZ gene incubated in serum-free DMEM for 24 h were stimulated with TPA (100 ng/ml) for 6 h in the absence (A) or presence (B) of IFN{gamma} treatment for 60 min or 24 h before stimulation with TPA (C).

 
Chronic exposure to IFN{gamma} does not impair other PKA-dependent nuclear effects
To begin to map the sites where IFN{gamma} interferes with cAMP signaling, we examined whether nuclear effects of PKA other than CRE-regulated gene expression were diminished in cells chronically exposed to IFN{gamma}. TSH stimulates the activity of nuclear (p85) and cytosolic (p70) isoforms of p70/p85s6k (33), a serine/threonine-specific protein kinase that regulates cell cycle progression and protein translation through effects on ribosomal protein S6 (reviewed in Refs. 34, 35). The effects of TSH on p70/p85s6k are cAMP mediated (33) and PKA dependent (36). The effects of IFN{gamma} effects on p70/p85s6k activity were examined in Western blotting experiments using an antibody directed to the phosphorylated form of ribosomal protein S6, its best known substrate. We previously demonstrated that the effects of TSH on S6 phosphorylation reflect effects on p70/p85s6k activity (33). After its addition to cells in basal medium, TSH and 8BrcAMP stimulated a 20-fold increase in S6 phosphorylation, indicative of p70/p85s6k activation (Fig. 6Go, S6-P). Whether added together (data not shown) or 48 h before stimulation, IFN{gamma} had no effect on TSH- or 8BrcAMP-stimulated S6 phosphorylation.



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  		Figure 6. Chronic IFN{gamma} treatment does not impair cAMP-stimulated p70/85S6k activity. WRT cells incubated for 48 h in basal medium in the absence (-) or presence (+) of IFN{gamma} (500 U/ml) were stimulated with TSH (1 mU/ml) or 8BrcAMP (1 mM) for 45 min. Cell lysates (60 µg) were analyzed by immunostaining with a phospho-specific S6 antibody (S6-P) or with an antibody raised to p70S6k/p85S6k (p85). Hyperphosphorylation, indicative of activation, was detected as a shift in the electrophoretic mobility of p85S6k. Three independent experiments yielded similar results.

 
To corroborate these findings, we examined the effects of IFN{gamma} on p85s6k, the nuclear isoform of p70/p85s6k. TSH, 8BrcAMP, and forskolin (data not shown) induced a shift in the electrophoretic mobility of p85s6k, indicative of phosphorylation. Importantly, chronic exposure to IFN{gamma} had no effect on the p85s6k mobility shift (Fig. 6Go, p85), indicating that IFN{gamma} does not impair all cAMP-regulated nuclear activities. To provide additional support for this idea, the effects of IFN{gamma} on CREB phosphorylation were investigated. PKA-mediated phosphorylation of serine 133 in CREB is required for CRE-lacZ gene expression. Microinjection of a CREB-specific antibody abolished CRE-regulated lacZ gene expression in several cell lines expressing this marker gene (37). The effects of IFN{gamma} on CREB phosphorylation were monitored in immunostaining assays using an antibody raised to the serine 133-phosphorylated form of CREB. TSH and 8BrcAMP stimulated CREB phosphorylation, as indicated by intense nuclear staining, and this was unaffected by pretreatment with IFN{gamma} for 48 h (Fig. 7Go). These results suggest that the inhibitory effects of IFN{gamma} on CRE-regulated gene expression lie distal to CREB phosphorylation.



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  		Figure 7. Chronic IFN{gamma} treatment does not impair CREB phosphorylation. WRT cells incubated for 48 h in basal medium in the absence (-) or presence (+) of IFN{gamma} (100 U/ml) were stimulated with TSH (1 mU/ml) or 8BrcAMP (1 mM) for 90 min. CREB phosphorylation was assessed by immunostaining with a phospho-specific CREB antibody. Two experiments, performed in duplicate, yielded similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the inhibitory effects of IFN{gamma} on thyroid-specific gene expression and proliferation are widely appreciated, the molecular mechanisms for these effects are largely unknown. In exponentially growing FRTL-5 cells, IFN{gamma} impaired expression of the TSHR through effects on TITF-1 DNA-binding activity (22). However, promoter constructs with deleted or mutated TITF-1-binding sites were still repressed by IFN{gamma}, indicating that there are additional targets of IFN{gamma}-mediated repression. In support of this view, we identified two promoter elements, AP-1 and CRE enhancers, subject to IFN{gamma}-mediated down-regulation in thyroid cells and fibroblasts.

IFN{gamma} has long been known to impair TSH effects on gene expression and proliferation. Despite this, in human (30) and rat thyroid cells (19), IFN{gamma} did not prevent cAMP accumulation in response to TSH, and in one case, it enhanced TSH effects on cAMP levels (20). These results implied that IFN{gamma} would not impair other acute effects stimulated by TSH. Indeed, we demonstrate for the first time that IFN{gamma} has no effect on TSH-stimulated p70/p85s6k activity and CRE-regulated gene expression when the two agents are added concomitantly. Even when added 60 min before TSH addition, IFN{gamma} did not impair CRE-regulated gene expression. In contrast, chronic exposure to IFN{gamma} for a minimum of 16 h before stimulation with TSH and 8BrcAMP led to a marked reduction in CRE-regulated gene expression, results that have not been previously reported. Despite this, nuclear p85s6k activity and CREB phosphorylation were not reduced after chronic exposure to IFN{gamma}, mapping the locus of inhibition to a site distal to nuclear PKA activity and CREB phosphorylation. These results agree with those reported in FRTL-5 cells, where treatment with IFN{gamma} did not affect protein complexes formed by CBP and the TSHR CRE element, although it impaired TSHR expression (22).

AP-1-regulated gene expression was also impaired after chronic exposure to IFN{gamma}. Unlike the CRE, however, gene expression from the AP-1 reporter was modestly reduced even when IFN{gamma} was added acutely. The ability of IFN{gamma} to impair AP-1-regulated genes after acute treatment and to repress CRE-regulated genes after chronic treatment argues for multiple mechanisms for IFN{gamma} effects. In macrophages, acute treatment with IFN{gamma} impaired AP-1-regulated promoters through competition between STAT1{alpha} and AP-1/ets domain transcription factors for limiting concentrations of CBP and p300 (32). Whether a similar mechanism is responsible for the inhibitory effects of acute IFN{gamma} on AP-1-regulated promoters in thyroid cells is not yet clear.

The chronic effects of IFN{gamma} on CRE promoter activity suggest that they are mediated through changes in gene expression. This hypothesis has not been directly tested, partly because IFN{gamma}-treated WRT cells die after treatment with protein synthesis inhibitors. However, in FRTL-5 cells, IFN{gamma} induces the expression of class II trans-activator. Similar to IFN{gamma}, overexpression of class II trans-activator increases the formation of a novel protein/DNA complex containing CBP on the human leukocyte antigen-DR{alpha} promoter (17). It is tempting to speculate that sequestration of CBP contributes to the inhibitory effects of IFN{gamma} on CRE-regulated genes in cells chronically exposed to IFN{gamma}. However, given the large number of genes whose expression is regulated by IFN{gamma}, there are likely to be many ways in which chronic exposure to IFN{gamma} interferes with CRE-regulated transcription.

Conflicting effects of IFN{gamma} on Tg expression have been reported in FRTL-5 cells. In an early report, IFN{gamma} down-regulated TSH-stimulated Tg message levels (28). In another report, IFN{gamma} alone had no effect on TSH-stimulated Tg or thyroid peroxidase message levels, although in combination with tumor necrosis factor-{alpha} both messenger RNAs were reduced (29). In human Graves’ thyrocytes, IFN{gamma} reduced TSH- and cAMP-stimulated Tg messenger RNA levels (30). Our results provide the first demonstration that IFN{gamma} reduces Tg protein levels stimulated by TSH. These effects were observed in cells arrested in medium supplemented only with BSA and then treated solely with TSH. This may be important given the differential effects of IFN{gamma} on Tg expression stimulated by TSH (where it decreases Tg expression) or serum (where it increases Tg expression). A precedent exists in the literature for confounding effects of variable medium components on thyroid cell proliferation and differentiation (38).

WRT cells resemble FRTL-5 and various human thyroid cell preparations in their response to IFN{gamma}, where this cytokine has been shown to inhibit proliferation (12, 19, 20), decrease Tg expression (23, 28, 29, 30) and secretion (5, 31), and up-regulate MHC class I expression (5, 10, 15). Unlike IFN{gamma}, IL-1ß did not impair TSH-induced Tg expression or DNA synthesis in WRT cells. These results differ somewhat from those reported in human cells, where IL-1ß impaired Tg expression stimulated by TSH (39, 40; reviewed in Ref. 41). It is noteworthy that although IL-1ß reduced iodide uptake in FRTL-5 cells, it had no effect on Tg secretion induced by TSH (42). In rat thyroid cells, conflicting results have been obtained regarding the effects of IL-1ß on DNA synthesis, where this cytokine has been reported to inhibit (43) or to have no effect (14) on TSH-stimulated DNA synthesis. Unlike these studies, which used recombinant human IL-1ß, our experiments were performed with rat rIL-1ß. Whether these differences reflect species-specific cytokine effects on rat thyroid cell growth is unknown, but has been discussed previously (14, 43).

Cross-talk between signaling pathways activated by TSH and IFN{gamma} may be important in thyroid cell pathophysiology. Although thyrocytes neither synthesize nor secrete IFN{gamma}, immune cells provide a local source of IFN{gamma} during infiltration of the thyroid gland. When used clinically, IFN induces thyroid dysfunction (44), perhaps as a consequence of its ability to reprogram thyroid-specific gene expression. To date, IFN{gamma} effects have been localized to effects on TITF-1 activity in the context of the TSHR promoter (22) and to the activation of a nuclear protein that binds to the human Tg promoter (23). The wide range of biological effects impaired by IFN{gamma} is difficult to reconcile with only these sites of action. Our data identify two specific promoter elements, CRE and AP-1 enhancers, that are also targets of IFN{gamma}-mediated repression. Although these elements are not directly involved in TSH regulation of Tg expression, CREB, in association with another DNA-binding protein, has been reported to bind to an incomplete CRE element present in the human Tg promoter (45). IFN{gamma}-mediated CRE promoter inhibition is not restricted to thyroid cells and therefore may be an important regulator of cAMP signaling in other cell types. Taken together, our findings suggest that IFN{gamma} contributes to thyroid dysfunction through acute inhibitory effects on AP-1-regulated gene expression as well as through chronic inhibitory effects on CRE-regulated genes.


   Acknowledgments
 
The authors thank Svetlana Savina for her technical assistance, and Dr. M. Birnbaum for the phospho-specific S6 antibody.


   Footnotes
 
1 This work was supported by National Research Service Award 5-F31-NS09883 from NINDS (to A.S.) and a K02 award (DK02494) from the NIDDK (to J.M.), and in part by the Lucille P. Markey Charitable Trust and the American Heart Association (9650551N). Back

Received August 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor. Vit Horm 50:287–384[Medline]
  2. Avvedimento V, Tramontano D, Ursini MV, Monticelli A, DiLauro RD 1984 The level of thyroglobulin mRNA is regulated by TSH both in vitro and in vivo. Biochem Biophys Res Commun 122:472–477[CrossRef][Medline]
  3. Ossendorp FA, Leer LM, Bruning PF, Brink JAM, Sterk A, Vijlder JJM 1989 Iodination of newly synthesized thyroglobulin by FRTL-5 cells is selective and thyrotropin dependent. Mol Cell Endocrinol 66:199–205[CrossRef][Medline]
  4. Heuverswyn BV, Streydio C, Brocas H, Refetoff S, Dumont J, Vassart G 1984 Thyrotropin controls transcription of the thyroglobulin gene. Proc Natl Acad Sci USA 81:5941–5945[Abstract/Free Full Text]
  5. Nagayama Y, Yamashita S, Hirayu H, Izumi M, Uga T, Ishikawa N, Ito K, Nagataki S 1989 Regulation of thyroid peroxidase and thyroglobulin gene expression by thyrotropin in cultured human thyroid cells. J Clin Endocrinol Metab 68:1155–1159[Abstract/Free Full Text]
  6. Magnusson RP, Rapoport B 1985 Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity. Endocrinology 116:1493–1500[Abstract/Free Full Text]
  7. Toyoda N, Nishikawa M, Mori Y, Gondou A, Ogawa Y, Yonemoto T, Yoshimura M, Masaki H, Inada M 1992 Thyrotropin and triiodothyronine regulate iodothyronine 5'-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells. Endocrinology 131:389–394[Abstract/Free Full Text]
  8. Dai G, Levy O, Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460[CrossRef][Medline]
  9. Levy O, Dai G, Riedel C, Ginter CS, Paul EM, Lebowitz AN, Carrasco N 1997 Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody. Proc Natl Acad Sci USA 94:5568–5573[Abstract/Free Full Text]
  10. Saji M, Moriarty J, Ban T, Singer DS, Kohn LD 1992 Major histocompatibility complex class I gene expression in rat thyroid cells is regulated by hormones, methimazole, and iodide as well as interferon. J Clin Endocrinol Metab 75:871–878[Abstract]
  11. Bottazzo GF, Pujol-Borrell R, Hanafusa T, Feldmann M 1983 Role of aberrant HLA-DR expression and antigen presentation in induction of thyroid autoimmunity. Lancet 2:1115–1119[Medline]
  12. Platzer M, Neufeld DS, Piccinini LA, Davies TF 1987 Induction of rat thyroid cell MHC class II antigen by thyrotropin and {gamma} interferon. Endocrinology 121:2087–2092[Abstract/Free Full Text]
  13. Piccinini LA, Roman SH, Davies TF 1987 Autoimmune thyroid disease and thyroid cell class II major histocompatibility complex antigens. Clin Endocrinol (Oxf) 26:253–272[Medline]
  14. Weetman AP, Rees AJ 1988 Synergistic effects of recombinant tumour necrosis factor and interferon-gamma on rat thyroid cell growth and Ia antigen expression. Immunology 63:285–289[Medline]
  15. Chiovato L, Lapi P, Mariotti S, Prete GD, Carli MD, Pinchera A 1994 Simultaneous expression of thyroid peroxidase and human leukocyte antigen-DR by human thyroid cells: modulation by thyrotropin, thyroid-stimulating antibody, and interferon-{gamma}. J Clin Endocrinol Metab 79:653–656[Abstract]
  16. Montani V, Shong M, Taniguchi S, Suzuki K, Giuliani C, Napolitano G, Saito J, Saji M, Fiorentino B, Reimold AM, Singer DS, Kohn LD 1998 Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocytes: opposite effects of inteferon and methimazole. Endocrinology 139:290–302[Abstract/Free Full Text]
  17. Montani V, Taniguchi S-I, Shong M, Suzuki K, Ohmore M, Giuliani C, Napolitano G, Saji M, Fiorentino B, Reimold AM, Ting JP-Y, Kohn LD, Singer DS 1998 Major histocompatibility class II HLA-DR{alpha} gene expression in thyrocytes: counter regulation by the class II transactivator and the thyroid Y box protein. Endocrinology 139:280–289[Abstract/Free Full Text]
  18. Kawakami A, Eguchi K, Matsuoka N, Tsuboi M, Kawabe Y, Ishikawa N, Ito K, Nagataki S 1996 Thyroid-stimulating hormone inhibits Fas antigen-mediated apoptosis of human thyrocytes in vitro. Endocrinology 137:3163–3169[Abstract]
  19. Misaki T, Tramontano D, Ingbar S 1988 Effect of rat gamma and non-{gamma} interferons on the expression of Ia antigen, growth, and differentiated functions of FRTL-5 cells. Endocrinology 123:2849–2855[Abstract/Free Full Text]
  20. Zakarija M, McKenzie JM 1989 Influence of cytokines on growth and differentiated function of FRTL5 cells. Endocrinology 125:1260–1265[Abstract/Free Full Text]
  21. Guazzi S, Price M, Felice MD, Damante G, Mattei MG, Lauro RD 1990 Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays novel DNA binding specificity. EMBO J 9:3631–3639[Medline]
  22. Ohe K, Ikuyama S, Takayanagi R, Kohn LD, Nawata H 1996 Interferon-{gamma} suppresses thyrotropin receptor promoter activity by reducing thyroid transcription factor-1 (TTF-1) binding to its recognition site. Mol Endocrinol 10:827–836
  23. Kung AWC, Lau KS 1998 Gamma-interferon activates a nuclear protein that binds to the {gamma}-interferon activation site of the thyroglobulin gene. J Mol Endocrinol 20:293–298[Abstract]
  24. Meinkoth J, Alberts AS, Feramisco JR 1990 Construction of mammalian cell lines with indicator genes driven by regulated promoters. CIBA Found Symp 150:47–56[Medline]
  25. Meinkoth JL, Goldsmith PK, Spiegel AM, Feramisco JR, Burrow GN 1992 Inhibition of TSH-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylyl cyclase Gs. J Biol Chem 267:13239–13245[Abstract/Free Full Text]
  26. Kupperman E, Wofford D, Wen W, Meinkoth JL 1996 Ras inhibits thyroglobulin expression but not cyclic adenosine monophosphate-mediated signaling in Wistar rat thyrocytes. Endocrinology 137:96–104[Abstract]
  27. Miller MJ, Rioux L, Prendergast GV, Cannon S, White MA, Meinkoth JL 1998 Differential effects of protein kinase A on ras effector pathways. Mol Cell Biol 18:3718–3726[Abstract/Free Full Text]
  28. Graves P, Neufeld DS, Davies TF 1989 Differential cytokine regulation of MHC class II and thyroglobulin mRNAs in rat thyroid cells. Mol Endocrinol 3:758–762[Abstract/Free Full Text]
  29. Tang K-T, Braverman LE, DeVito WJ 1996 Tumor necrosis factor-{alpha} and interferon-{gamma} modulate gene expression of type I 5'-deiodinase, thyroid peroxidase, and thyroglobulin in FRTL-5 rat thyroid cells. Endocrinology 136:881–888[Abstract]
  30. Kung AWC, Lau KS 1990 Interferon-{gamma} inhibits thyrotropin-induced thyroglobulin gene transcription in cultured human thyrocytes. J Clin Endocrinol Metab 70:1512–1517[Abstract/Free Full Text]
  31. Huber GK, Davies TF 1990 Human fetal thyroid cell growth in vitro: system characterization and cytokine inhibition. Endocrinology 126:869–875[Abstract/Free Full Text]
  32. Horvai AE, Xu L, Korzus E, Brard G, Kalafus D, Mullen T, Rose DW, Rosenfeld MG, Glass CK 1997 Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300. Proc Natl Acad Sci USA 94:1074–1079[Abstract/Free Full Text]
  33. Cass LA, Meinkoth JL 1998 Differential effects of cyclic adenosine 3', 5'-monophosphate on p70 ribosomal S6 kinase. Endocrinology 139:1991–1998[Abstract/Free Full Text]
  34. Chou MM, Blenis J 1995 The 70 kDa S6 kinase: regulation of a kinase with multiple roles in mitogenic signalling. Curr Opin Cell Biol 7:806–814[CrossRef][Medline]
  35. Pullen N, Thomas G 1997 The modular phosphorylation and activation of p70s6k. FEBS Lett 410:78–82[CrossRef][Medline]
  36. Cass LA, Summers SA, Prendergast GV, Backer JM, Birnbaum MJ, Meinkoth JL 1999 PKA-dependent and -independent signaling pathways contribute to cAMP-stimulated proliferation. Mol Cell Biol 19:5882–5891[Abstract/Free Full Text]
  37. Meinkoth JL, Montminy MR, Fink JS, Feramisco JR 1991 Induction of a cyclic AMP-responsive gene in living cells requires the nuclear factor CREB. Mol Cell Biol 11:1759–1764[Abstract/Free Full Text]
  38. Zakarija M, McKenzie JM 1989 Variations in the culture medium for FRTL-5 cells: effects on growth and iodide uptake. Endocrinology 125:1253–1259[Abstract/Free Full Text]
  39. Yamashita S, Kimura H, Ahizawa K, Nagayama Y, Hirayu H, Izumi M, Nagataki S 1988 Interleukin-1 inhibits thyrotropin-induced human thyroglobulin gene expression. J Endocrinol 122:177–183[CrossRef]
  40. Kung AWC, Lau KS 1990 Interleukin-1ß modulates thyrotropin-induced thyroglobulin mRNA transcription through 3',5'-cyclic adenosine monophosphate. Endocrinology 127:1369–1374[Abstract/Free Full Text]
  41. Rasmussen AK, Feldt-Rasmussen U, Bendtzen K 1993 The effect of interleukin-1ß on the thyroid gland. Autoimmunity 16:141–148[Medline]
  42. Reimers JI, Rasmussen AK, Karlsen AE, Bjerre U, Liang H, Morin O, Andersen HU, Mandrup-Poulsen T, Burger AG, Feldt-Rasmussen U, Nerup J 1996 Interleukin-1ß inhibits rat thyroid cell function in vivo and in vitro by an NO-independent mechanism and induces hypothyroidism and accelerated thyroiditis in diabetes-prone BB rats. J Endocrinol 151:147–157[Abstract/Free Full Text]
  43. Rasmussen AK, Kayser L, Bech K, Feldt-Rasmussen, Perrild H, Bendtzen K 1990 Differential effects of interleukin 1{alpha} and 1ß on cultured human and rat thyroid epithelial cells. Acta Endocrinol (Copenh) 122:520–526[Abstract/Free Full Text]
  44. Miossec P 1997 Cytokine-induced autoimmune disorders. Drug Safety 17:93–104[Medline]
  45. Berg V, Vassart G, Christophe D 1997 A zinc-dependent DNA-binding activity co-operates with cAMP-responsive-element-binding protein to activate the human thyroglobulin enhancer. Biochem J 323:349–357
  46. Dumont JE, Lamy F, Roger P, Maenhaut C 1992 Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol Rev 72:667–697[Free Full Text]



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