Transforming Growth Factor-ß1 Down-Regulation of Major Histocompatibility Complex Class I in Thyrocytes: Coordinate Regulation Of Two Separate Elements by Thyroid-Specific as Well as Ubiquitous Transcription Factors

Giorgio Napolitano, Valeria Montani, Cesidio Giuliani, Simonetta Di Vincenzo, Ines Bucci, Valentina Todisco, Giovanna Laglia, Anna Coppa, Dinah S. Singer, Minoru Nakazato, Leonard D. Kohn, Giulia Colletta and Fabrizio Monaco

Chair of Endocrinology (G.N., V.M., C.G., S.D.V., I.B., V.T., G.L., F.M.) Department of Medicine and Department of Oncology and Neuroscience (G.C.) University "G. D’Annunzio", Chieti, Italy 66100
Department of Experimental Medicine and Pathology (A.C.) University "La Spienza", Rome, Italy 00161
Experimental Immunology Branch (D.S.S.) National Cancer Institute and Metabolic Diseases Branch (M.N., L.D.K.) National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, Maryland 20892-1800


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transforming growth factor (TGF)-ß1-decreased major histocompatibility complex (MHC) class I gene expression in thyrocytes is transcriptional; it involves trans factors and cis elements important for hormone- as well as iodide-regulated thyroid growth and function. Thus, in rat FRTL-5 thyrocytes, TGF-ß1 regulates two elements within -203 bp of the transcription start site of the MHC class I 5'-flanking region: Enhancer A, -180 to -170 bp, and a downstream regulatory element (DRE), -127 to -90 bp, that contains a cAMP response element (CRE)-like sequence. TGF-ß1 reduces the interaction of a NF-{kappa}B p50/fra-2 heterodimer (MOD-1) with Enhancer A while increasing its interaction with a NF-{kappa}B p50/p65 heterodimer. Both reduced MOD-1 and increased p50/p65 suppresses class I expression. Decreased MOD-1 and increased p50/p65 have been separately associated with the ability of autoregulatory (high) concentrations of iodide to suppress thyrocyte growth and function, as well as MHC class I expression. TGF-ß1 has two effects on the downstream regulatory element (DRE). It increases DRE binding of a ubiquitously expressed Y-box protein, termed TSEP-1 (TSHR suppressor element binding protein-1) in rat thyroid cells; TSEP-1 has been shown separately to be an important suppressor of the TSH receptor (TSHR) in addition to MHC class I and class II expression. It also decreases the binding of a thyroid-specific trans factor, thyroid transcription factor-1 (TTF-1), to the DRE, reflecting the ability of TGF-ß1 to decrease TTF-1 RNA levels. TGF-ß1-decreased TTF-1 expression accounts in part for TGF-ß1-decreased thyroid growth and function, since decreased TTF-1 has been shown to decrease thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR gene expression, coincident with decreased MHC class I. Finally, we show that TGF-ß1 increases c-jun RNA levels and induces the formation of new complexes involving c-jun, fra-2, ATF-1, and c-fos, which react with Enhancer A and the DRE. TGF-ß1 effects on c-jun may be a pivotal fulcrum in the hitherto unrecognized coordinate regulation of Enhancer A and the DRE.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transforming growth factor-ß (TGF-ß) polypeptides regulate the growth, function, and immune properties of cells, decreasing, for example, major histocompatibility complex (MHC) class I and class II expression basally or after {gamma}-interferon (IFN) stimulation (1, 2, 3, 4). In thyrocytes, including functioning rat FRTL-5 cells in continuous culture, TGF-ß1 inhibits cell proliferation and TSH-induced iodide uptake, thyroglobulin (TG) biosynthesis, and endothelin production (5, 6, 7, 8). The role of TGF-ß polypeptides in regulating MHC gene expression in the thyroid is less clear; however, TGF-ß1-deficient transgenic mice have increased MHC class I and II levels in many organs and develop a rapid, wasting, immune disease (9, 10, 11).

Abnormal expression of MHC Class I and II is associated with thyroid autoimmunity (12, 13). Recent work has shown that aberrant class II, together with abnormal TSH receptor (TSHR) expression, can induce autoimmune hyperthyroidism and a Graves’-like syndrome in mice, despite a normal immune system (14). TGF-ß1-induced immune disease does not develop in class II-deficient animals (15), and TGF-ß1 regulation of class II expression has been linked to conserved proximal elements of the 5'-flanking region (16). Abnormal class I expression is also linked to Graves’ disease, since class I suppression has been shown to be an important component of the immunosuppressive action of high (autoregulatory) concentrations of iodide or of methimazole, which are used to treat Graves’ disease (13, 17, 18, 19, 20, 21). The mechanism of TGF-ß1 regulation of MHC class I in thyrocytes is unknown as is the relationship of such regulation to TGF-ß1-inhibited thyroid growth and function.

Evidence has accumulated that hormones and growth factors that regulate FRTL-5 cell growth and function can coordinately decrease MHC class I expression and may prevent autoimmune thyroid disease (13, 17, 22, 23, 24, 25). Coordinate regulation of class I expression and thyroid genes important for cell growth and function has been shown to result from the interaction of thyroid-restricted and ubiquitous transcription factors with common cis-elements on the 5'-flanking regions of the class I and thyroid-restricted genes such as the TSHR (13, 22, 23, 24, 25). It was of interest, therefore, to document the effect of TGF-ß1 on thyrocyte MHC class I expression and relate this action to the regulation of thyroid growth and function in the FRTL-5 thyrocyte by hormones and other factors.

In this report, we show that TGF-ß1 decreases MHC class I expression in FRTL-5 thyroid cells and acts transcriptionally within -203 bp of the start site of the gene. It regulates the interaction of thyroid-restricted as well as ubiquitous transcription factors with two cis elements, Enhancer A, -180 to -170 bp, and a downstream regulatory element (DRE), -127 to -90 bp, whose activity requires a cAMP response element (CRE)-like sequence, -107 to -100 bp (13, 23, 24, 25). These same cis elements and/or trans factors are involved in hormone and iodide control of genes important for thyroid growth and function, i.e. the TSHR, TG, thyroid peroxidase (TPO), and the sodium iodide symporter (NIS).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TGF-ß1 Down-Regulates MHC Class I Transcription in Thyrocytes; Its Action Involves Enhancer A and a Downstream CRE-Containing Regulatory Element (DRE), -127 to -90 bp
TGF-ß1 significantly reduces MHC class I RNA levels in FRTL-5 rat thyroid cells (Fig. 1AGo). The mean decrease in four separate experiments, when compared with ß-actin RNA levels (which were unaffected by TGF-ß1), was maximal between 5 and 10 ng/ml, evident within 6 h, optimal by 24, and nonexistent by 72 h (Table 1Go). The class I RNA decrease was accompanied by a decrease in class I antigen expression in control or {gamma}-IFN-treated cells (Fig. 1BGo). TGF-ß1 had no effect on cAMP levels in the cells (data not shown) and its effect was evident in non-TSH treated cells where cAMP levels are low (Table 1Go). We could not distinguish an immediate early response to TGF-ß1 from a later, secondary response based on these data.



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Figure 1. Effect of TGF-ß1 on Class I mRNA Levels (A) and on Class I Antigen Expression (B)

FRTL-5 cells were grown to 60% confluency. In panel A, cells were exposed to 5 ng/ml TGF-ß1 for 12 h, at which time total RNA was isolated and Northern analysis performed using class I and ß-actin probes. A representative blot is presented as is the mean class I/ß actin ratio ± SD from four independent experiments. Control values are arbitrarily set at 1; the decrease by TGF-ß1 is significant at P < 0.05. In panel B, class I antigen levels were measured by fluorescence-activated cell sorting (FACS) analysis in control cells (left) or cells treated with 100 U/ml {gamma}-interferon for 24 h (right) and in the presence or absence of 5 ng/ml TGF-ß1 for the last 12 h. The dashed line represents the Leu-4 background control. Data are representative of four experiments with similar results.

 

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Table 1. Time and Concentration Effect of TGF-ß1 on MHC Class I RNA Levels and on Promoter Activity in FRTL-5 Cells Stably Transfected with the p(-203)Class I-CAT Chimera

 
The TGF-ß1-induced decrease in class I RNA levels and antigen expression reflects a predominantly transcriptional action. Thus, TGF-ß1 reduced the activity of Class I promoter/chloramphenicol acetyl transferase (CAT) chimeras in transiently (Fig. 2AGo) or stably transfected FRTL-5 cells (Tables 1Go and 2Go). The TGF-ß1 effect was dependent on the TGF-ß1 concentration and the time of treatment, in a manner similar to the effect of TGF-ß1 on class I RNA levels (Table 1Go). The effect on transcription was evident whether cells were maintained in the presence or absence of TSH (Table 2Go).



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Figure 2. Effect of TGF-ß1 on the Promoter Activity of Class I CAT Chimeras Having Different 5'-Extensions

In panel A, FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then shifted again to 6H for 20–24 h. Cells were washed, incubated 1 h with 20 µg of the class I-CAT chimera plasmid DNA, 2 µg pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H medium for 40–48 h, and then maintained therein another 12 h with or without 5 ng/ml TGF-ß1. CAT activity was measured at that time and normalized for transfection efficiency. Cell viability was approximately 89 ± 4% in four separate experiments; data are the mean of these experiments, performed in triplicate, ± SD. The class I chimeras were PD1 MHC promoter/CAT constructs having different 5'-lengths as noted. In panel B, a diagrammatic representation of the different class I chimeras is presented. Some of the different regulatory elements in each are noted: a, the tissue-specific region; b, Enhancer A (Enh A); c, IRE; d, the 38-bp DRE (24 ); e, the CRE-like site (24 ); f, the CAAT box. pSV0 is the control vector.

 

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Table 2. Effect of 5 ng/ml TGF-ß1 on MHC Class I Promoter Activity in FRTL-5 Cells Stably Transfected with Class I-CAT Chimeras Having Different Lengths of the Class I 5'-Flanking Region

 
Initial experiments localized the site of the TGF-ß1-induced class I decrease within -127 bp of the start of transcription. Thus, similar decreases in promoter activity were measured in both transiently (Fig. 2AGo) and stably (Table 2Go) transfected FRTL-5 cells containing 5'-deletions of an 1100-bp Class I promoter/CAT chimera containing 549 [p(-549)CAT], 203 [p(-203)CAT], and 127 [p(-127)CAT] bp of 5'-flanking sequence from the start of transcription. However, TGF-ß1 activity was markedly decreased in a p(-56)CAT chimera containing 56 bp of 5'-flanking sequence and was not measurable in the promoterless pSV0 control CAT construct (Fig. 2AGo and Table 2Go).

Although the 5'-deletion data could not distinguish a difference in the TGF-ß1-induced decrease in p(-127)CAT and p(-203)CAT promoter activities because of the error values, involvement of elements upstream of, as well as within -127 bp, of 5'-flanking sequence became evident when we mutated individual elements in the p(-203)CAT chimera: 203MA, 203{Delta}CRE, or both together, 203MA{Delta}CRE (Fig. 3AGo). Thus, when the activity of the p(-203) wild-type CAT chimera was compared with the activity of a -203-bp chimera having the Enhancer A element (-180 to -170 bp) mutated (p203MA), the Enhancer A mutation caused a significant, but incomplete, decrease in the TGF-ß1 response (Fig. 3Go). This was not the case if the IFN response element (IRE; -161 to -150 bp) was deleted (Fig. 3Go; p203{Delta}IRE), i.e. the effect of mutating Enhancer A seemed specific.



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Figure 3. Effect of TGF-ß1 (A) on Deletion or Substitution Mutants of Enhancer A, the IRE, or the CRE in the p(-203)class I-CAT Constructs That Are Diagrammatically Noted in Panel B

Individually isolated clones stably transfected with the wild-type p(-203) bp class I-CAT chimera or with the p(-203)class I chimera containing the noted mutation of Enhancer A [p(-203)MA], a deletion of the IRE [p(-203){Delta}IRE], a deletion of the CRE-like sequence [p(-203){Delta}CRE], or both a mutation of Enhancer A plus a deletion of the CRE [p(-203)MA{Delta}CRE] were grown to 70% confluency in 6H medium and then maintained without TSH (5H medium) for 6 days. Cells were then exposed to 5 ng/ml concentrations of TGF-ß1 for 12 h before CAT activity was measured. Data are the mean of three experiments, performed in triplicate, ± SD. A star denotes a significant P < 0.05 decrease vs. its untreated control, which has arbitrarily been set at 100%. Statistical differences between constructs, P < 0.05, are also noted. The sequences of the CRE, the IRE, Enhancer A, and mutated Enhancer A are noted on the bottom.

 
Within -127 bp, the TGF-ß1 response was decreased, but not completely, if the CRE-like sequence (-107 to -100 bp) within the DRE (26), -127 to -90 bp, was deleted (Fig. 3Go; p203{Delta}CRE). It appeared, however, that Enhancer A and the CRE might be functionally interrelated, since modification of both was required to abolish TGF-ß1 activity completely (Fig. 3Go; p203MA{Delta}CRE).

We conclude that TGF-ß1 down-regulates MHC class I transcriptionally and that its effect involves at least two elements, Enhancer A and the CRE-like site. The remainder of this report focuses on the effect of TGF-ß1 on factors interacting with Enhancer A or the CRE-like sequence, on the relationship of these elements and factors, and on the basis for their relationships. Since the TGF-ß1 decrease in class I promoter activity was near maximal 12 h after treatment with 5 ng/ml TGF-ß1, these conditions were used in all subsequent experiments unless otherwise noted.

The TGF-ß1 Action on Enhancer A Involves Coordinately Decreased Binding of a fra-2/NF-{kappa}B p50 Subunit Heterodimer and Increased Binding of a p50/p65 NF-{kappa}B Subunit Heterodimer; Both Actions Suppress MHC Class I Gene Expression
Using electrophoretic mobility shift assay (EMSA) and a 74-bp radiolabeled probe spanning -203 to -130 bp, which includes both Enhancer A and the IRE (Fig. 4Go, bottom), we performed binding studies under low salt conditions. Cell extracts from TGF-ß1-treated cells exhibited a significant reduction in a complex that migrated near the top of the gel (Fig. 4AGo, lane 2 vs. 3, arrow) and had characteristics of MOD-1 (23, 25). MOD-1 is a complex between enhancer A and a heterodimer of fra-2 with the p50 subunit of NF-{kappa}B (23, 25). MOD-1 is decreased by hydrocortisone (23), by high concentrations of iodide (25), and by phorbol esters (25), all of which regulate FRTL-5 cell growth and function (25, 26, 27, 28, 29, 30). A decrease in MOD-1 has been circumstantially associated with decreased class I expression, whereas its increase, i.e. by {gamma}-IFN, is associated with increased class I expression (23, 25).



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Figure 4. Ability of TGF-ß1 to Decrease Formation of the MOD-1 Protein/DNA Complex between FRTL-5 Thyroid Cell Extracts and Enhancer A (A) but Increase Formation of a p50/p65 Heterodimer Complex with Enhancer A (B)

FRTL-5 thyroid cells were grown to near confluency in 6H medium containing 5% calf serum and then maintained for 6 days in 5H medium (no TSH) with 5% calf serum. Cells were fed fresh medium with or without 5 ng/ml TGF-ß1 and extracts prepared after 12 h. In panel A, cell extracts were incubated with a radiolabeled 74-bp fragment of the MHC class I promoter between -203 and -130 bp, termed the 74 probe and diagrammatically represented in panel C. Incubations were in a low salt buffer without detergents; EMSAs were used to identify protein DNA complexes. Lane 1 contains the radioactive probe alone; lanes 2 and 3 are, respectively, incubations with radioactive probe and extracts of cells treated with 5 ng/ml TGF-ß1 or from control cells. In lanes 4 and 5 the incubations with control cells included a 100-fold excess, over labeled probe, of unlabeled oligonucleotide with the sequence of mutated Enhancer A or native Enhancer A, respectively (see Fig. 3Go for sequences). The MOD-1 complex is noted by the arrow and was defined by its ability to be inhibited and supershifted with antibodies to the p50 subunit of NF-{kappa}B and to fra-2 but not control antibodies (23 25 ). In panel B, nuclear extracts were incubated with the radiolabeled oligonucleotide containing the Enhancer A sequence of the class I promoter (see Fig. 3Go, bottom); incubations were in a high-salt buffer containing detergent, rather than the low-salt buffer without detergent used in panel A. Lane 1 is the radioactive probe alone; lanes 2 and 3 are, respectively, the incubations with radioactive probe and extracts of control cells or cells treated with 5 ng/ml TGF-ß1. Lanes 4 and 5 represent incubations with extracts from cells treated with TGF-ß1 but containing unlabeled oligonucleotide competitors with the wild-type or mutant Enhancer A, each in a 100-fold excess. Lane 6 notes the effect of an unlabeled oligonucleotide with the consensus NF-{kappa}B binding site, also in a 100-fold excess over probe. Lanes 7–14 depict the effect of serum from a normal rabbit or rabbit polyclonal antibodies to the noted transcription factors. Arrows denote the location of the TGF-ß1-induced complex identified as a p50/p65 heterodimer based on the antibody results and the location of a complex that we suggest is a p50 homodimer based 1) on the antisera data and 2) on its mobility on a gel relative to complexes formed by different concentrations of authentic p50 protein (23 25 ).

 
To establish that this complex was MOD-1 and interacted with Enhancer A, we first showed that formation of the complex was decreased by a 100-fold excess of an oligonucleotide with the sequence of Enhancer A, but not by the same concentration of the oligonucleotide with a mutated Enhancer A sequence (Fig. 4AGo, lane 5 vs. 4). The mutated Enhancer A sequence has been separately shown to lose Enhancer A function and MOD-1 binding (23, 25). Additionally, we showed that formation of the complex was inhibited and/or supershifted by specific antibodies against fra-2 or the p50 subunit of NF{kappa}B, but not by antibodies against fra-1, the p65 subunit of NF{kappa}B, or other c-fos family members [data not shown but exactly as previously described (23, 25)]. We could, therefore, unequivocally identify this TGF-ß1-decreased complex as MOD-1.

When binding to Enhancer A was simultaneously performed in high-salt conditions containing detergents, rather than low-salt conditions, nuclear extracts from TGF-ß1- treated FRTL-5 cells exhibited increased binding of a p50/p65 heterodimer of NF-{kappa}B to Enhancer A (Fig. 4BGo, lane 3 vs. 2), coordinately with decreased MOD-1 binding (Fig. 4AGo). Thus, formation of the TGF-ß1-increased complex was prevented by unlabeled Enhancer A (100x), but not by mutant Enhancer A at the same concentration (Fig. 4BGo, lanes 4 and 5) and was decreased and/or supershifted by antibodies to the p50 and p65 subunits of NF-{kappa}B (Fig. 4BGo, lanes 8 and 9, respectively), but not by antibodies to c-rel, fra-1, fra-2, c-fos, or c-jun (Fig. 4BGo, lanes 10–14). Similar increases in the p50/p65 heterodimer are induced by iodide and phorbol esters (25).

The complex was decreased by the unlabeled consensus sequence of NF-{kappa}B, 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Fig. 4BGo, lanes 6), which contains the core GGGGA sequence of enhancer A, 5'-TGGGGAGTCCCCGTG-3', but differs in having a longer inverted repeat (italicized) and a different spacing to the inverted repeat. Thus, the GGGGA is a critical element in the formation of the TGF-ß1-induced p50/p65 heterodimer complex, just as it is for the iodide- or phorbol ester-increased p50/p65 heterodimer (25).

The decrease in MOD 1 and the increase in p50/p65 heterodimer is not associated with an ability of TGF-ß1 to affect a change in I{kappa}B in the cytosol or localization of p65 molecules in the nucleus at early times (Table 3Go). Thus, fractionation and Western blot analysis revealed no significant decrease in I{kappa}B or increase in p65 molecules in the nuclear extract until after 12 h (Table 3Go). Changes in both, however, may contribute at 24 h.


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Table 3. Effect of TGF-ß1 on the Localization of p65 Molecules in the Nucleus as a Function of Time

 
To unequivocally establish the functional relevance of the binding of MOD-1 or the p50/p65 heterodimer to Enhancer A, we transiently cotransfected the -203-bp class I-CAT chimera with full length cDNAs encoding the NF-{kappa}B p65 subunit (pMT2T-p65), the NF-{kappa}B p50 subunit (pMT2T-p50), fra-2 (pRSV- fra-2), combinations thereof, or their control vectors (pMT2T and pRSV). Cotransfection of p50 plus fra-2 markedly increased the promoter activity of the p(-203) class I/CAT chimera (Fig. 5Go, second open bar). This was not true of their respective control vectors (Fig. 5Go, first open bar), of p65 plus fra-2, of either alone, or of their control vectors (Fig. 5Go). In contrast, cotransfection of p50 plus p65 significantly decreased the promoter activity of the p(-203) class I/CAT chimera (Fig. 5Go, fifth open bar). The ability of TGF-ß1 to decrease class I expression was maintained or relatively enhanced in the p50/fra-2 cotransfected cells (Fig. 5Go, second black bar) but was lost in the p50/p65 transfected cells (Fig. 5Go, fifth black bar). Transfections involving p203MA exhibited no responses to cotransfections with p50/fra-2 or p50/p65 (data not shown), unequivocally linking these effects to Enhancer A. Additionally, the effect of TGF-ß1 on the p50/fra-2 and p50/p65 cotransfections was very specific for the wild-type p(203) promoter.



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Figure 5. Effect of TGF-ß1 on the Transient Expression of the p(-203) Class I-CAT Chimera That Was Cotransfected with cDNAs Encoding the p50 or p65 Subunits of NF-{kappa}B, fra-2, or Control Vectors Containing These cDNAs, pMT2T and pRSV, Respectively

FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then shifted again to 6H for 20–24 h. Cells were washed, incubated 1 h with 20 µg class I-CAT chimera plasmid DNA, 10 µg of each of the expression vectors, pMT2T and pRSV, with or without the inserted cDNAs for p50, p65, and fra-2, 2 µg pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H medium for 40–48 h, and then maintained therein another 12 h with or without 5 ng/ml TGF-ß1. CAT activity was measured at that time and normalized for transfection efficiency. Data are the mean of four separate experiments, performed in triplicate ± SD. Cell viability was approximately 88 ± 5%. *, Significant P < 0.05 decrease induced by TGF-ß1 treatment by comparison to the control cells, which have not been treated with TGF-ß1. **, A significant decrease in control activity, P < 0.05, by comparison to cells transfected with p(-203)CAT plus pMT2T and pRSV control vectors (first open bar) and whose activity is arbitrarily set at 100%. ***, A significant increase, P < 0.02, by comparison to cells transfected with p(-203)CAT plus pMT2T and pRSV control vectors (first open bar) and whose activity is arbitrarily set at 100%. ****, A significant P < 0.01 decrease induced by TGF-ß1 by comparison to an untreated control.

 
In sum, the data are the first direct demonstration of the functional importance of MOD-1 and the p50/p65 heterodimer on class I expression, of the ability of TGF-ß1 to concurrently decrease their binding to Enhancer A, and of their importance to the TGF-ß1-down-regulatory effect on class I. Additional p65 alone, in cells transfected with pTMT- p65, does not significantly decrease class I gene expression or enhance TGF-ß1 suppressive activity, suggesting there is no significant free p50 to form new p50/p65 heterodimer complexes with enhancer A. The increase in p50/p65 heterodimer may result from the decrease in MOD-1 complex, releasing p50 already within the nucleus to interact with p65. The fra-2 would, in this scenario, be freed to form new complexes; this will be evidenced below. The loss of the TGF-ß1 suppressive action in p50/p65 transfected cells suggests that p50/p65 suppression is a dominant effect of TGF-ß1 on Enhancer A.

TGF-ß1 Decreases the Binding of a Tissue-Specific Transcription Factor (TTF-1) to the DRE, but Increases the Binding of a Ubiquitous Transcription Factor (TSEP-1, a Y-box Protein); These Are, Respectively, an Enhancer and Suppressor of Class I Gene Expression Whose Activity Requires the CRE-Like Site
TSH and forskolin down-regulate class I gene expression by their action on the DRE, -127 to -90 bp, whose function depends on the CRE-like sequence, -107 to -100 bp (24). TGF-ß1-induced down-regulation of class I also requires the CRE-like site (Fig. 3Go). TSH/forskolin-decreased class I expression is associated with their ability to induce the formation of new complexes with a class I probe containing 127 bp of 5'-flanking region (-127 to +1 bp) (24). These complexes reflect the loss or gain, respectively, of binding to the DRE by thyroid transcription factor-1 (TTF-1) and the murine homolog of the human Y-box protein, YB-1, which we had cloned and termed TSHR suppressor element binding protein-1 (TSEP-1) (24, 31). TTF-1 and TSEP-1 are tissue-specific and ubiquitous transcription factors, respectively; they are also, respectively, an enhancer and suppressor of class I gene expression (24).

TGF-ß1-treatment of FRTL-5 cells maintained without TSH (in 5H medium) results in the increase of two complexes with the radiolabeled -127 bp probe (Fig. 6AGo, lane 2 vs. 3, Complexes A and B) that have the same migratory properties as those increased by TSH (Fig. 6AGo, lane 4 vs. 3). TGF-ß1-treatment of FRTL-5 cells maintained with TSH (6H medium) results in a third new complex termed complex C, but no increase in complexes A and B relative to TGF-ß1-treatment without TSH (Fig. 6AGo, lane 5 vs. lane 2). Formation of the C complex is prevented by including a 150-fold excess of unlabeled oligonucleotide with a consensus AP-1 site and is inhibited by including anti-c-jun in the incubation (Fig. 6CGo, lanes 2 and 3, respectively, vs. lane 1).



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Figure 6. Ability of TGF-ß1 to Alter the Binding of Proteins to the Class I DRE, -127 to -90 bp, Whose Activity Is Dependent on the CRE-Like Site, -107 to -100 bp

FRTL-5 thyroid cells were grown to near-confluency in 6H medium containing 5% calf serum and then maintained for 6 days in 5H medium (no TSH) with 5% calf serum. Cells were fed fresh 5H or 6H medium for 24 h and then treated with or without 5 ng/ml TGF-ß1 for 12 h. Whole-cell extracts were prepared as described (Materials and Methods). In panel A, cell extracts were incubated with a radiolabeled probe encompassing -127 to +1 bp of the 5'-flanking region of the class I promoter, termed the -127 probe and diagrammatically represented at the bottom of the panel. The DRE, -127 to -90 bp, and its encompassed CRE-like site, -107 to -100 bp, is noted; its properties were previously characterized (24 ). Lane 1 contains probe alone, lanes 2 and 3 are incubations containing probe plus extracts from cells without TSH, lanes 4 and 5 are plus extracts from cells maintained with TSH. Lanes 3 and 4 are control cell extracts; lanes 2 and 5 are extracts from cells treated with TGF-ß1. A and B denote complexes increased by TGF-ß1 or TSH treatment. C denotes a complex induced by TGF-ß1 in cells also treated with TSH. In panel B, EMSAs depict results from incubations with extracts from cells maintained without TSH but treated with 5 ng/ml TGF-ß1 for 12 h. Incubations in this experiment included a 100-fold excess of unlabeled oligonucleotides representing different elements: CRE-1, the DRE from -127 to -90 bp (lane 1); the CRE octamer, -107 to -100 bp, plus 6 bp on either side (lane 2); oligonucleotide C, the TTF-1/Pax-8 binding site from the TG promoter (lane 4); and the TSEP-1 binding site from the TSHR promoter (lane 5). Panel C depicts the effect of including a consensus AP-1 oligonucleotide (lane 2) or an antiserum to c-jun (Santa Cruz Biotechnology, Inc.) in incubations with the radiolabeled -127 probe plus extract from cells treated with 5 ng/ml TGF-ß1 plus TSH. The C complex is noted.

 
The similarity of the migration of the A and B complexes formed by the -127 probe with extracts of TSH or TGF-ß1-treated cells, and the absence of a significant increase in the intensity of the complexes formed by extracts from cells treated with both TSH and TGF-ß1, suggested that the complexes were related and were likely to involve the DRE, -127 to -90 bp, as previously described (24). That the A and B complexes in the TGF-ß1-treated cell extracts involved the DRE, rather than the CRE site alone, was evidenced by the ability of an oligonucleotide with the sequence of the class I promoter from -120 to -90 bp (termed CRE-1) to inhibit formation of the TGF-ß1-induced A and B complexes, but not a homolog that contains only the CRE octamer, -107 to -100 bp, plus 6 bp on its 3'- or 5'-ends (Fig. 6BGo, lanes 1 and 2, respectively, vs. 3). An oligonucleotide containing CRE-1, with the CRE-like site, -107 to -100 bp, deleted ({Delta}CRE), also did not compete (data not shown). These data indicated that the TGF-ß1-induced A and B complexes, like the TSH/cAMP-induced A and B complexes (24), involved binding to the DRE, but also required the CRE-like site for this binding, consistent with the loss of the TGF-ß1-activity in p203{Delta}CRE, (Fig. 3AGo).

EMSA revealed that TSEP-1 was a critical protein in the formation of the TGF-ß1-induced, as well as the TSH/cAMP-induced A and B complex (Fig. 6BGo). Thus, a single-strand oligonucleotide containing one of the TSEP-1 binding sites of the TSHR (34), 5'-AAACTACCTCTCAACGCATCCG-3' (-216 to -190 bp in the TSHR 5'-flanking region) inhibited formation of the TGF-ß1-induced A and B complexes (Fig. 6BGo, lane 5 vs. 3). Similar inhibition was seen using a different single-strand Y-box binding site on the TSHR, -162 to -140 bp; and no inhibition was evident using a single-strand oligonucleotide with a mutation in the Y-box protein binding element, 5'-AAACTAGTCTTCAACGCATCCG-3' (italicized and bold), which had been shown to lose TSEP-1 binding activity (31) (data not shown). No inhibition was evident using a double-strand oligonucleotide containing the Pax-8/TTF-1 binding site of the TG promoter, termed oligo C (32) (Fig. 6BGo, lane 4 vs. 3), or a single-strand oligonucleotide with the sequence of the upstream SSBP-1/TTF-1 binding site of the TSHR, 5'-CTTGTTGCACGGTGAATTCACGAGAAG-3', -886 to -858 bp in the TSHR 5'-flanking region (33). SSBP-1, Pax-8, and TTF-1 have been shown to interact with the DRE as well as TSEP-1 (24).

To verify the functional relevance of TSEP-1 for TGF-ß1 action, we transiently cotransfected the -203-bp class I CAT chimera with pRc/CMV-TSEP-1 or its control vector pRc/CMV (Fig. 7Go). Overexpression of TSEP-1 cDNA suppressed p(-203)CAT activity (Fig. 7Go, last open bar), and the ability of TGF-ß1 to decrease p(-203)CAT activity was lost (Fig. 7Go, last black bar vs. last open bar). Overexpression of the control vector had no effect on activity with or without TGF-ß1.



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Figure 7. Effect of TGF-ß1 on Transient Expression of the p(-203) class I-CAT Chimera That Was Cotransfected with a cDNAs Encoding pTSEP-1 cDNA or Its Control Vector, pRc/CMV

FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then shifted back to 6H medium for 24 h. Cells were washed, incubated 1 h with 20 µg of the p(-203) class I-CAT chimera plasmid DNA, 10 µg of each of the pRc/CMV vectors with or without the full-length TSEP-1 insert (31 ), 2 µg pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H medium for 12 h, in 5H medium for 40 h, and then with or without 5 ng/ml TGF-ß1 for 12 h. CAT activity was measured and normalized for transfection efficiency. Data are the mean of four separate experiments, performed in triplicate ± SD. Cell viability was approximately 89 ±6%. *, A significant P < 0.05 decrease induced by TGF-ß1 treatment by comparison to the control cells, which have not been treated with TGF-ß1. **, A loss in the ability of TGF-ß1 to cause a significant decrease by comparison to control activity as well as a significant decrease, P < 0.05, in the p(-203)CAT control by comparison to cells transfected with p(-203)CAT ± the pRc/CMV control vectors.

 
Although the EMSA experiment using the -127-bp probe and competition with oligo C (Fig. 6BGo, lane 4) did not indicate that TTF-1 was involved in the effect of TGF-ß1 on the DRE, separate results did suggest TTF-1 involvement (Fig. 8Go). Thus, TGF-ß1 treatment, for the same time and at the same concentration that was near maximally effective in decreasing class I expression (12 h at 5 ng/ml), significantly (P < 0.05) decreased TTF-1 RNA and protein levels (Fig. 8AGo). TTF-1 protein was measured by the ability of TGF-ß1 treatment to decrease TTF-1 complex formation between nuclear extracts and the radiolabeled TSHR TTF-1 site probe (Fig. 8AGo, top), which is TTF-1 specific (33, 34). It was confirmed by the ability of anti-TTF-1 to completely supershift the complexes in the control and TGF-ß1-treated extracts (data not shown).



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Figure 8. Effect of TGF-ß1 on TTF-1 RNA levels and TTF-1 Protein Binding to a TTF-1- Specific Binding Site from the TSHR (A), as Well as Its Effect on TTF-1-Increased p(-203) Class I-CAT Chimera Activity

In panels A and B, FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then treated with 5 ng/ml TGF-ß1 for 12 h. In panel A, total RNA was isolated and Northern analysis performed using TTF-1 and ß-actin probes. A representative blot is presented (top, RNA), as is the mean TTF-1/ß actin ratio ± SD from four independent experiments (bottom). Control values for the latter data were arbitrarily set at 1; *, a significant, P < 0.05, decrease. In panel A, nuclear extracts were also isolated from the cells, incubated with a radiolabeled TTF-1-specific probe from the TSHR (top, protein), and TTF-1 binding measured by EMSA (34 ). The TSHR TTF-1 element was used in this experiment because it is a pure TTF-1 binding element which does not interact with Pax-8 (34 ). The class I DRE has two TTF-1 sites, one upstream and one downstream of the CRE (Ref. 24 and footnote 1). The upstream TTF-1 site, like oligo C from the TG promoter, also binds Pax-8 (Ref. 24 and footnote 1). We could not use the pure downstream class I TTF-1 site because the functional binding site overlaps the CRE and would give ambiguous results. The TSHR TTF-1 site is well spaced from the CRE. The specificity of the complex was established by incubating each with anti-TTF-1, which eliminated and supershifted the complex (data not shown). In panel B, FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then shifted back to 6H medium for 24 h. Cells were washed, incubated 1 h with 20 µg of the p(-203) class I-CAT chimera plasmid DNA, 10 µg of each of the pRc/CMV vectors with or without the full length TTF-1 (34 ), 2 µg pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H medium for 12 h, in 5H medium for 40 h, and then with or without 5 ng/ml TGF-ß1 for 12 h. CAT activity was measured and normalized for transfection efficiency. Data are the mean of four separate experiments, performed in triplicate ± SD. Cell viability was approximately 91 ± 4%. *, A significant P < 0.02 decrease induced by TGF-ß1 treatment by comparison to the control cells, which have not been treated with TGF-ß1 (first set); **, (last set) denotes a significant decrease of P < 0.05. In the middle set, ** denotes a significant increase in control activity, P < 0.02, by comparison to cells transfected with p(-203)CAT alone; and *** denotes a significant (P < 0.01) TGF-ß1-induced decrease in class I promoter activity by comparison to the control cells that were transfected with p(-203)CAT plus pRc/CMV-TTF-1.

 
Consistent with earlier studies (24), when we transiently cotransfected the -203-bp class I CAT chimera with pRc/CMV-TTF-1 or its control vector pRc/CMV (Fig. 8BGo), overexpression of TTF-1 cDNA increased p(-203)CAT activity. Overexpression of TTF-1 resulted in the near-complete loss of TGF-ß1 suppression (Fig. 8BGo). Overexpression of the control vector had no effect on activity with or without TGF-ß1. In sum, TGF-ß1 also appears to decrease expression and binding of a tissue-specific enhancer, TTF-1, to the DRE, -127 to -90 bp, as well as increase the binding of a ubiquitous suppressor, a Y-box protein. The net result is to suppress the promoter by modulating the activity of the DRE.

The Two TGF-ß1-Responsive Areas, Enhancer A and the CRE-Containing DRE, Appear to be Linked by the Action of TGF-ß1 on c-jun Protein
We explored the possibility that the effects of TGF-ß1 on Enhancer A and the DRE were interrelated, since modification of both elements appeared to be required for maximal loss of TGF-ß1 activity (Fig. 3Go). Using the radiolabeled 74-bp probe encompassing Enhancer A, we performed competition experiments with a 100-fold excess of unlabeled oligonucleotides encompassing the DRE or reacting with trans factors binding to the element (24): CRE-1, the element from -127 to -90 bp (24); the CRE octamer plus 6 bp on either side (-113 to -94 bp); oligonucleotide C, the TTF-1/Pax-8 binding site from the TG promoter (32); and the TSEP-1 binding site from the TSHR promoter between -162 and -140 bp (31) (Fig. 9AGo). Conversely, we used a 100-fold excess of unlabeled oligonucleotides with the sequence of Enhancer A or the mutated form of Enhancer A to compete for complexes formed with the radiolabeled -127 probe encompassing the DRE (Fig. 9BGo).



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Figure 9. MOD-1 Binding in the Presence of Oligonucleotide Competitors of Protein/DNA Complexes with DRE (A) and the Converse (B), the Effect of Enhancer A as a Competitor of the Binding of Complexes to the DRE

FRTL-5 thyroid cells were grown to near confluency in 6H medium and then maintained for 6 days in 5H medium (no TSH). Cells were fed fresh 5H medium for 24 h and maintained with or without 5 ng/ml TGF-ß1 for 12 h, and whole cell extracts were prepared for EMSA. In panel A, cell extracts from cells maintained without TSH and untreated with TGF-ß1 were incubated with a radiolabeled 74-bp probe from -203 to -130 bp, which includes Enhancer A. Lane 1 contains an incubation with no unlabeled competitor as a control; however, other incubations included a 100-fold excess of the following unlabeled oligonucleotides: the TSEP-1 binding site from the TSHR promoter (lane 2); CRE-1, the element from -127 to -90 bp, which includes the CRE-like site from -107 to -100 bp (lane 3); oligonucleotide C, the TTF-1/Pax-8 binding site from the TG promoter (lane 4); and the CRE octamer, -107 to -100 bp, plus 6 bp on either side (lane 5). The MOD-1 complex is noted by the arrow and was defined by its ability to be inhibited and supershifted with antibodies to the p50 subunit of NF-{kappa}B and to fra-2 but not control antibodies (data not shown). In panel B, cell extracts from cells maintained without TSH and untreated with TGF-ß1 (lanes 1–3) were incubated with a radiolabeled probe encompassing -127 to +1 bp of the 5'-flanking region of the class I promoter, termed the -127 probe. Lane 1 contains no competitor; lanes 2 and 3 are incubations containing a 100-fold excess of unlabeled Enhancer A or unlabeled mutated Enhancer A oligonucleotide (see Fig. 3BGo, bottom). Lane 4 contains extract from cells treated with TSH plus TGF-ß1, lane 5 from cells maintained without TSH but treated with TGF-ß1. Complex C, which is increased by TGF-ß1 treatment of cells maintained with TSH (lane 4), is noted by an arrow.

 
Oligo TSEP-1 (Fig. 9AGo, lane 2) and oligo CRE-1 (Fig. 9AGo, lane 3) inhibited the formation of MOD-1 with the 74-bp probe, but not oligo C (lane 4) or the CRE octamer plus 6 bp on either side (lane 5). Using the same extracts with the -127 probe, an excess of an oligonucleotide with the sequence of Enhancer A, but not mutated Enhancer A, induced the formation of a complex migrating with the same mobility as the C complex induced by TGF-ß1 in cells maintained with TSH (Fig. 9BGo, lane 2 vs. 4). We concluded, therefore, that the two TGF-ß1-sensitive regions, the DRE and Enhancer A, were not only functionally interrelated (Fig. 3Go), but also related by interactions involving common trans factors (Fig. 9Go). We wondered whether this relationship involved c-jun, since complex C appeared to be related to c-jun (Fig. 6CGo). TGF-ß1 can increase c-jun levels and function in fibroblasts and other cells (35, 36, 37).

TGF-ß1 treatment of FRTL-5 cells increased c-jun RNA levels (Fig. 10Go). A significant increase (35 ± 7%) was measurable within 1 h; the maximal increase was evident by 3–5 h and continued through 12 h (Fig. 10Go). The increase was not evident at 18 h. TGF-ß1-increased c-jun RNA was associated, therefore, with TGF-ß1-induced decreases in class I gene expression at early times.



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Figure 10. Effect of TGF-ß1 on c-jun RNA Levels

FRTL-5 cells were grown to 60% confluency in medium with TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and then treated with 5 ng/ml TGF-ß1 for 12 h. Total RNA was isolated and Northern analysis performed using c-jun and ß-actin probes (Materials and Methods). A representative blot is presented; the same results were obtained in four separate experiments.

 
We then incubated the -127 radiolabeled probe with a c-jun homodimer, AP-1, in the absence or presence of extracts from cells maintained without TSH (Fig. 11Go). Neither recombinant AP-1 nor a control recombinant protein that was similarly prepared, SSBP-1 (33), was able to bind to the radiolabeled, double-stranded -127-bp probe in the absence of extract (Fig. 11Go, lanes 2 and 3, respectively). However, in the presence of extract from cells maintained in the absence of TSH and never treated with TGF-ß1, we could observe the appearance of the C complex in the presence of the c-jun dimer, AP-1, but not SSBP-1 (Fig. 11Go, lanes 5 and 6, respectively). This suggested c-jun might indeed be relevant to the TGF-ß1-induced C complex in cells treated with TGF-ß1 as well as TSH (Fig. 6AGo, lane 5).



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Figure 11. Recombinant AP-1 (c-jun) Modifies the EMSA Pattern of Protein/DNA Complexes Formed by the DRE

Recombinant AP-1 or SSBP-1 was incubated with the radiolabeled -127 probe either alone (lanes 2–3) or with extracts (lanes 5–6) from cells maintained without TSH. A new complex is formed with recombinant AP-1 plus extract, which has the mobility of the C complex interacting with the DRE (Figs. 6Go and 9Go).

 
Complexes other than the complex C are increased by the addition of recombinant AP-1; however, in part, this seems to be an effect of added protein since the increases in these other complexes are induced by recombinant SSBP-1 (Fig. 11Go, lane 6) or albumin (data not shown). The increase in the upper complex mimics that observed when unlabeled enhancer A is added to reactions with the -127 probe plus extract from cells that are not TSH stimulated (Fig. 9BGo, lane 2). We speculate that increased c-jun, added exogenously (Fig. 11Go) or endogenously by competition with Enhancer A (Fig. 9BGo, lane 2; Fig. 13Go, model below) will increase proteins that are components of the upper complex, i.e. activating transcription factor-1 (ATF-1) adducts (Ref. 24 ; see below). We have no alternative explanation for this phenomenon at this time.



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Figure 13. Diagramatic Representation of the TGF-ß1 Effect on Factors Interacting with the Tissue-Specific and Hormone-Sensitive Regions of the MHC Class I Promoter

The TGF-ß1 effect on c-jun is hypothesized to be a key component in altering the make up and interactions of the different complexes existing in the basal state. See text for description.

 
In addition, we used a radiolabeled oligonucleotide containing the AP-1 consensus binding site as a probe to measure complexes formed in extracts from cells maintained without TSH and either treated or not treated with TGF-ß1 (Fig. 12Go). Extracts from cells that had not been treated with TGF-ß1 had a single major complex in the presence of the radiolabeled AP-1 consensus binding site (Fig. 12AGo, lane 2, Complex X). Extracts from cells treated with TGF-ß1 formed a prominent, additional, slower migrating complex with the radiolabeled AP-1 consensus binding site (Fig. 12AGo, lane 3, Complex Y). Using specific antibodies, we showed that formation of the slower migrating Y complex was nearly completely inhibited by anti-c-jun (Fig. 12Go, lane 9). It was also nearly completely inhibited by anti-c-fos (Fig. 12AGo, lane 8), significantly decreased by anti-ATF-1 (Fig. 12AGo, lane 7), decreased by anti-fra-2 (Fig. 12AGo, lane 4), but not at all decreased by antibodies to the p50 and p65 subunits of NF-{kappa}B (Fig. 12AGo, lanes 5 and 6). In contrast, the faster moving, lower X complex, which was constitutive in the absence of TGF-ß1 treatment of the cells (Fig. 12AGo, lane 2), was predominantly inhibited by anti-ATF-1 (Fig. 12AGo, lane 7) and much less dramatically by anti-c-fos or anti-c-jun (Fig. 12AGo, lanes 8 and 9). These data established that TGF-ß1 treatment increased c-jun RNA and c-jun binding activity; they established that the increase in c-jun protein was associated with heterodimers of ATF-1, c-fos, and fra-2.



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Figure 12. The Effect of TGF-ß1 on Protein/DNA Complexes Formed with a Radiolabeled Oligonucleotide with the Sequence of the Consensus AP-1 Binding Site

FRTL-5 cells were grown to near confluency in 6H medium and then maintained 6 days in 5H medium with no TSH. Cells were fed fresh 5H medium for 24 h and maintained with or without 5 ng/ml TGF-ß1 for 12 h, and whole-cell extracts were prepared for EMSA. In panel A, cell extracts from cells maintained without TSH and treated (lane 3) or not (lane 2) with TGF-ß1 were incubated with a radiolabeled consensus AP-1 probe; probe alone is in lane 1. Lanes 4–9 contain extracts from cells maintained without TSH and treated with TGF-ß1, which were preincubated with the noted antibodies before addition of the radiolabeled probe. In panel B, the extracts from cells maintained without TSH but treated with TGF-ß1 were preincubated with a 100-fold excess of unlabeled oligonucleotides containing the following sequences: the CRE octamer, -107 to -100 bp, plus 6 bp on either side (lane 1); CRE-1, the DRE, -127 to -90 bp, which includes the CRE-like site from -107 to -100 bp (lane 2); and Enhancer A. In Panel C, the complex formed between AP-1 (c-jun) protein and the radiolabeled oligonucleotide containing the consensus AP-1 site (lane 1) is compared with the complex in extracts from cells maintained without TSH and TGF-ß1 (lane 2) and a new complex formed when AP-1 (c-jun) is incubated with the AP-1 consensus binding site plus the extract from cells maintained without TSH and TGF-ß1 (lane 3). This gel was run for a greater length of time and longer distance to allow the different complexes to be clearly depicted. The X and Y complexes were the same in all panels based on parallel incubations with anti-ATF-1, anti-c-fos, and anti-c-jun as in panel A.

 
That this phenomenon was relevant to complexes formed with Enhancer A and the DRE was evident in two experiments. First, in competition experiments, an unlabeled oligonucleotide with the sequence of the DRE or of Enhancer A could each inhibit formation of both complexes formed with the AP-1 consensus site (Fig. 12BGo, lanes 2 and 3, respectively). This was not true of the attenuated CRE-site oligo, -113 to -94 bp (Fig. 12BGo, lane 1), which does not prevent formation of TGF-ß1-induced complexes with the -127 probe. Thus, both Enhancer A and the DRE interacted with the complexes involving c-jun, because they competed for the complexes that interacted with the consensus AP-1 oligonucleotide; their availability for AP-1 complex formation was enhanced by TGF-ß1. It should be recalled that in the absence of TGF-ß1 or TSH/cAMP, the MHC class I CRE, as well as the TSHR CRE, normally form complexes with ATF-1/CREB (cAMP response element binding protein) (24, 38, 39, 40).

Second, it was noted that addition of AP-1 protein to an extract from cells maintained without TGF-ß1 (Fig. 12CGo, lane 3) resulted in a complex with the AP-1 consensus binding sequence that migrated not only more slowly than the AP-1 (c-jun) protein alone (Fig. 12CGo, lane 1), but also with a mobility similar to the upper C complex in the extracts from the TGF-ß1-treated cells, i.e. a phenomenon that is mimicked by TGF-ß1 treatment, which induces the formation of complexes involving c-jun, c-fos, ATF-1, and fra-2.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Abnormal MHC class I expression has been implicated in autoimmune thyroid disease (12, 13, 17, 18, 19, 20, 21), whereas TGF-ß1-mediated suppression of MHC class I has been implicated as a means to mitigate autoimmune disease (1, 2, 3, 4, 9, 10, 11). The present study shows that TGF-ß1 decreases MHC class I RNA levels and antigen expression in thyrocytes; more importantly, it addresses the mechanism by which TGF-ß1 down-regulates MHC class I. We show that the TGF-ß1 suppression of class I is largely transcriptional and that its effects involve at least two distinct sequences located within -203 bp of the start of transcription. We show that the factors and elements involved in TGF-ß1 regulation of MHC class I are also involved in the hormone regulation of thyroid-specific genes important for thyroid growth and function.

The 5'-flanking region of the class I gene has a "hormone-sensitive region" (-203 to +1 bp) (13, 23, 24, 25, 41) and a "tissue- specific region" (-771 to -679 bp) (13, 41, 42, 43). Whereas the tissue-specific region controls constitutive class I expression in different cells (41, 42, 43), the hormone-sensitive region (HSR) is modulated by cytokines, growth factors, drugs, and hormones that regulate thyroid cell growth and function (13, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Among these, hydrocortisone (23), iodide (25), phorbol esters (25), and TSH (10, 17, 24) down-regulate class I expression, whereas IFN increases expression (13, 17, 23). The HSR is composed of several regulatory sequences, including Enhancer A (-180 to -170 bp), the IFN-response element or IRE (-161 to -150 bp), and the DRE, -127 to -90 bp, which contains a CRE-like site, -107 to -100 bp (13, 17, 23, 24, 25).

We show that TGF-ß1 acts like iodide or phorbol esters to decrease MOD-1 and increase p50/p65 heterodimer binding to Enhancer A. We provide transfection data using p50, p65, and fra-2 cDNAs to unequivocally show that MOD-1 increases, whereas the p50/p65 heterodimer decreases, class I activity. TGF-ß1 thus modulates transcription factor binding to Enhancer A the same as iodide or phorbol esters (23, 25), which control thyroid function and growth (23, 25, 26, 27, 28, 29, 30).

The DRE, -127 to -90 bp, whose activity is dependent on a CRE-like site (-107 to -100 bp), is the main site of action for TSH/cAMP repression (24). TSH/cAMP decreases formation of a CREB/ATF-1 complex, which binds to the CRE-like sequence (Ref. 24 and M. Shong, S. I. Taniguchi, G. Napolitano, M. Saji, M. Ohmori, M. Ohta, H. Shimura, K. Suzuki, D. S. Singer, and L. D. Kohn, in preparation). They decrease formation of a complex with a thyroid-restricted transcription factor, TTF-1, which recognizes sequences flanking each side of the CRE-like element (Ref. 24 and M. Shong, S. I. Taniguchi, G. Napolitano, M. Saji, M. Ohmori, M. Ohta, H. Shimura, K. Suzuki, D. S. Singer, and L. D. Kohn, in preparation). TSH/cAMP also coordinately increases the ability of a Y-box protein, TSHR suppressor element protein-1, TSEP-1 (31), to bind the DRE (24). Since TSEP-1 has a suppressive effect, whereas CREB/ATF-1 and TTF-1 are enhancers, the net effect of the TSH/cAMP action is a reduction in class I expression (24). We show that TGF-ß1 regulates the DRE by similarly modulating the binding of these same factors to the DRE: TSEP-1, TTF-1, and, as a result of increased c-jun/ATF-1 interactions, very likely CREB/ATF-1.

One feature of TGF-ß1 action that is not evident in previous studies with TSH, hydrocortisone, iodide, or phorbol esters appears to be the interactive involvement of both elements and the induction of a new complex, complex C, which we uncovered in studies of TGF-ß1-treated cells that were also maintained with TSH. The mutual interaction of the two elements is established by cross-competition experiments involving the ability of oligonucleotides with the sequence of one element to inhibit the binding properties of the other. We provide evidence that complex C involves c-jun and show that TGF-ß1 increases c-jun RNA and protein binding. We provide evidence that TGF-ß1 action increases c-jun interactions with ATF-1, c-fos, and fra-2 in EMSA studies using an AP-1 consensus binding site and that the new complexes interact with both elements, Enhancer A and the DRE. The effect of TGF-ß1 to increase c-jun protein levels has been reported in keratinocytes and fibroblasts (36). c-jun has also been reported to decrease MHC class I promoter activity at an insulin/serum-modulated element upstream (35) of the HSR studied herein.

Figure 13Go represents a hypothesis of the dynamic TGF-ß1 regulation of the hormone-responsive region of the -203 bp 5'-flanking region of class I. Since TGF-ß1 does not decrease I{kappa}B or increase the entrance of NF-{kappa}B subunits into the nucleus at 12 h, when the effect of TGF-ß1 is already near maximal, we speculate that TGF-ß1-increased c-jun is a pivotal fulcrum in the relationships between Enhancer A and the DRE (Fig. 13Go). We speculate that TGF-ß1-increased c-jun sets off an interlocking cascade of reactions that modulates the basal interactions of Enhancer A, the DRE, and the upstream silencer/enhancer in the tissue-specific region, -771 to -679 bp, since the latter can bind c-jun (G. Napolitano, M. Saji, C. Giuliani, S. I. Taniguchi, M. Shong, V. Montani, K. Suzuki, J. Weissman, D. S. Singer, and L. D. Kohn, manuscript in preparation). The increased c-jun RNA and binding activity alter the basal equilibrium favoring the formation of the MOD-1 (fra-2/p50) complex with Enhancer A, and instead favor the formation of a new complex involving c-jun and fra-2, thereby also increasing the available activated p50 subunit. We speculate that increased c-jun simultaneously may reduce the interaction of c-fos and p65 with the upstream silencer/enhancer in the tissue-specific region controlling class I expression (Fig. 13Go and G. Napolitano, M. Saji, C. Giuliani, S. I. Taniguchi, M. Shong, V. Montani, K. Suzuki, J. Weissman, D. S. Singer, and L. D. Kohn, manuscript in preparation. This would contribute c-fos to the c-jun/fra-2 complex and also increase available p65. The available p50 and p65 from these actions would allow formation of a p50/p65 heterodimer able to interact with Enhancer A and suppress class I expression (Fig. 13Go). We suggest that the c-jun/fra-2/c-fos complex might replace the CREB interaction with ATF-1 and its interaction with the DRE; CREB/ATF-1 binding to the DRE functions as a class I silencer in FRTL-5 cells (Refs. 26, 44 and G. Napolitano, M. Saji, C. Giuliani, S. I. Taniguchi, M. Shong, V. Montani, K. Suzuki, J. Weissman, D. S. Singer, and L. D. Kohn, manuscript in preparation) (Fig. 13Go). TGF-ß1 additionally increases TSEP-1, but decreases TTF-1 binding to the DRE (Fig. 13Go), thereby increasing the activity of a suppressor (TSEP-1) and decreasing the activity of an enhancer (TTF-1). The sum of actions decreases the binding of enhancers of class I expression (MOD-1, CREB/ATF-1, TTF-1) but increases the binding of suppressors (p50/p65, Y-box). Both types of modifications contribute to down-regulation of class I expression.

The Smad family of proteins are mediators of TGF-ß signaling (2, 37, 44, 45, 46, 47, 48). Complexes involving Smad2, Smad3, or Smad 4 can bind directly to DNA and act as transcription factors (44, 45, 46). Smad binding element (SBE) consensus sequences have been identified; concatemerization of these sequences confers Smad 3/4 and TGF-ß responsiveness. Smads can cooperate with other transcription factors and/or can be mediated by interactions with FAST-1 (2, 37, 44, 45, 46, 47). Smad transcriptional activity can also be regulated by binding to coactivators (p300/CBP, AP-1) or corepressors (Sin3a, Sky, TGIF) (2, 48). The presence of Smad2, Smad3, and Smad4 and their translocation into the nucleus in response to TGF-ß has been demonstrated in porcine thyroid cells (49). Smad2 and Smad4 are expressed in FRTL-5 cells and are functionally active in enhancing transcription (50).

The ability of TGF-ß1 to alter c-jun levels or binding activity may be mediated by its effects on Smad proteins and binding elements (37, 51). Smad proteins can regulate ATF/CREB and NF-{kappa}B family members (37, 53, 54). However, Smad-independent paths exist (55). Additionally, the basis for the ability of TGF-ß1 to decrease TTF-1 and increase/or activate TSEP-1 is unclear. Smad/Fast-1 and SBEs remain to be identified as mediators of TGF-ß effects on these transcription factors. The ability of TGF-ß1 to decrease TTF-1 levels, as shown by Northern analysis, may separately involve redox regulation. TGF-ß1 has been reported to have an oxidant effect (56), which can down-regulate TTF-1 binding to DNA (57). Given the complexity of the Smad system, it is premature to speculate what role they have in the regulation described herein. The relationship between Smad proteins and regulation by CREB, c-jun, ATF family members, or NF-{kappa}B will be complex and remains to be characterized in this system.

Independent of the role of Smad proteins, which is under investigation, our observations are consistent with previous studies. TGF-ß1 has been shown to modulate CREB proteins and ATF-1 (58, 59), c-jun family proteins (36, 37, 60, 61), and c-fos family proteins, including fra-2 (61). The coordinated action and physical interactions of transcription factors from different families have been widely demonstrated. Indeed, the ATF-1 protein has been reported to interact with both AP-1 as well as CRE consensus sequences (60). AP-1 has, in turn, been reported to interact with both c-fos family members (61) and NF-{kappa}B subunits (62). Such interactions are dependent on structural properties of the proteins involved; however, homologies between the binding sequences may favor the interaction. Thus, the CRE-like sequence (TGACGCGA) is similar to AP-1 consensus binding sequence (TGAc/gTCA) whose core is also present within the enhancer A region (TGGGGAGTCCCCGTG) and is similar to the NF-{kappa}B consensus sequence (GGGGACTTTTCCCC).

In sum, we show that TGF-ß1 decreases MHC class I expression by regulating trans factors interacting with Enhancer A and the DRE. The TGF-ß1-induced decrease in TTF-1 will decrease TG, TPO, NIS, and TSHR gene expression (22). The TGF-ß1 effects on MOD-1 and the p50/p65 heterodimer, like autoregulatory concentrations of iodide (25), will suppress thyroid growth and function (27, 28, 29, 30). TSEP-1 is a suppressor of TSHR expression (22, 31); increased TSEP-1 activity would, therefore, also decrease thyroid growth and function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Human Platelet TGF-ß1 was from Sigma (St. Louis, MO); [{alpha}-32P]deoxy-CTP (3000 Ci/mmol), [14C]chloramphenicol (50 mCi/mmol), and [{gamma}-32P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech (Arlington Heights, IL). Antibodies against the p50 and p65 subunits of NF-{kappa}B, the c-fos family, the ATF/CREB family, and c-jun were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiserum and control preimmune serum for rat TTF-1 were obtained from Dr. S. Kimura (National Cancer Institute, NIH, Bethesda, MD). The AP-1 consensus binding oligo was from Santa Cruz Biotechnology, Inc. The source of all other materials has been detailed (17, 23, 24, 25) or was Sigma.

The TTF-1 expression vector, pRc/CMV-TTF-1, was kindly provided by Dr. R. Di Lauro (Stazione Zoologica A. Dohrn, Villa Comunale, Naples, Italy). The pRc/CMV-TSEP-1 was constructed by ligating its full-length coding sequence with the pRc/CMV vector (Invitrogen, San Diego, CA) (31). The expression vectors pMT2T-p65 and pMT2T-p50 were kindly donated by Dr. Ulrich Siebenlist (NIAID, NIH, Bethesda, MD) and the expression vector, pRSV-fra-2, by Dr. Hideo Iba (University of Tokyo, Tokyo, Japan).

Cell Culture
FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore MD; ATCC No. CRL 8305) were a fresh subclone (F1) that had all properties previously detailed (13, 17, 22, 23, 24, 25, 26, 31, 33, 34, 38, 39, 40, 63, 64). Their doubling time with TSH was 36 ± 6 h; they were diploid and between their 5th and 25th passage. Cells were grown in 6H medium consisting of Coon’s modified F12 (Sigma) supplemented with 5% calf serum, 1 mM nonessential amino acids (Life Technologies, Inc., Gaithersburg, MD) and a mixture of six hormones: bovine TSH (1 x 10-10 M), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml) (63, 64). Fresh medium was added every 2 or 3 days, and cells were passaged every 7–10 days. In different experiments, as noted, cells were maintained in 5H medium which contains no TSH.

RNA Isolation and Northern Analysis
mRNA was isolated using a commercial kit (Quickprep mRNA Purification Kit, Pharmacia Biotech, Uppsala, Sweden), and Northern analysis was performed as described (17, 23, 24, 25). Hybridization was with 1.0 x 106 cpm/ml of the following cDNA probes: a 1.0-kb HpaI fragment of the MHC class I (17); ß-actin (kindly provided by Dr. B. Paterson, National Cancer Institute, NIH, Bethesda, MD); and a 926-bp HindIII-ApaI TTF-1 cDNA fragment excised from the pRc/CMV-TTF-1 vector (34). The c-jun probe was from Oncor (Gaithersburg, MD).

Construction of MHC Class I Promoter-CAT Chimeric Plasmids
Chloramphenicol acetyltransferase (CAT) chimeras of the MHC class I swine (PD1) 5'-flanking region have been described (23, 24, 25) and are inserted into the multicloning site of the pSV0-based CAT construct used as a control in all experiments. They are numbered from the nucleotide at the 5'-end to +1 bp, the start of transcription. CAT constructs with mutated MHC class I sequences were created by two-step recombinant PCR methods (65) as detailed (23, 24, 25). The sequences of all constructs were confirmed by a standard method (66).

Transfection and CAT Assay
Transient transfection used the Class I promoter/CAT chimeras and a diethylaminoethyl (DEAE)-dextran procedure (67). Cells were grown to 60% confluency in 6H medium, shifted to 5H medium for 7 days, and then shifted again to 6H for 20–24 h. Cells were washed twice with PBS, pH 7.4, and incubated 1 h with 5 ml serum-free medium without hormones (0H), containing 20 µg class I-CAT chimera plasmid DNA, 2 µg pRSV-luciferase, which was used to measure the efficiency of transfection (23, 24, 25), and 250 µg DEAE-dextran. Cells were then exposed to 10% dimethylsulfoxide in PBS for 3 min, washed twice in PBS, cultured in 6H medium for 40–48 h, and maintained therein another 12 h with or without TGF-ß1 as noted. Cell viability was approximately 80% in all experiments. Overexpression experiments involving cotransfection with pRc/CMV-TTF-1, pRc/CMV-TSEP-1, pMT2T-p65, pMT2T-p50, pRSV-fra-2, or their control vectors, pRc/CMV, pMT2T, and pRSV, included 10 µg of the appropriate plasmid DNA.

In some experiments we used FRTL-5 cells that had been stably transfected with the pSV0-based CAT-PD1 chimeras as described (23). To test the effect of TGF-ß1, three individually isolated clones of each construct were grown to 70% confluency in 6H medium, maintained without TSH (5H medium) for 6 days, and exposed to TGF-ß1 before CAT activity was measured. CAT assays were performed as described (23, 24, 25).

Cell and Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium until 60% confluent, and then maintained in 5H medium with 5% calf serum for 7 days, and finally exposed to TGF-ß1. Cellular extracts were prepared by a modification of methods described previously (23, 24, 68). In brief, cells were washed twice in cold PBS, pH 7.4, scraped, and centrifuged (500 x g). The cell pellet was resuspended in 2 volumes of Dignam buffer C (25% glycerol, 20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.5 mM dithiothreitol, 1 mg/ml leupeptin, 1 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride). The final NaCl concentration was adjusted on the basis of cell pellet volume to 0.42 M. Cells were lysed by repeated cycles of freezing and thawing. The extracts were centrifuged (100,000 x g) at 4 C for 20 min. The supernatant was recovered, aliquoted, and stored at -70 C.

Nuclear extracts were prepared as described previously (23, 24, 25, 31, 33, 34) from identically treated and harvested FRTL-5 cells. After centrifugation at 500 x g, the cells were suspended in 5 pellet volumes of 0.3 M sucrose and 2% Tween 40 in Buffer A [10 mM HEPES-KOH, pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). After freezing, thawing, and gently homogenizing, nuclei were isolated by centrifugation at 25,000 x g on a 1.5 M sucrose cushion containing the same buffer. Nuclei were lysed in Buffer B (10 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). After centrifugation at 100,000 x g for 1 h, the supernatant was dialyzed for use in gel mobility shift analyses using 10 mM Tris-Cl at pH 7.9, 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol, aliquoted, and stored at -70 C.

Electrophoretic Mobility Shift Assays (EMSAs)
DNA probes were created by restriction enzyme treatment of the chimeric CAT constructs described above and purified from 2% agarose gel using QIAEX (QIAGEN, Chatsworth, CA) (23, 24, 25, 31, 33, 34). Oligonucleotides were synthesized by Operon Technologies (Alameda, CA). They were labeled with [{alpha}-32P]dCTP using Klenow or with [{gamma}-32P]ATP using T4 polynucleotide kinase, and then purified on an 8% native polyacrylamide gel (23, 24, 25, 31, 33, 34).

EMSAs were performed as previously described (23, 25). Binding reactions in low salts and no detergent included 1.5 fmol [32P]DNA, 3 µg cell extract, and 1 µg poly(dI-dC) in 10 mM Tris-Cl, pH 7.9, 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol in a 20 µl total volume (23, 25). Binding reactions in high salts plus detergent included 1.5 fmol of [32P]DNA, 2 µg extract, and 0.5 µg poly(dI-dC) in 10 mM Tris-Cl, pH 7.9, 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol (25). Incubations were at room temperature for 30 min. Where indicated, unlabeled oligonucleotide competitors, recombinant proteins, or antibodies were added to the binding reaction and incubated with the extract for 20 min before the addition of labeled DNA. After incubations, reaction mixtures were electrophoresed on 4–5% native polyacrylamide gels at 160 V in 0.5xTBE at room temperature. Gels were dried and autoradiographed.

Determination of Effects of TGF-ß1 on I{kappa}B and NF-{kappa}B Localization and Levels
Measurements of I{kappa}B in the cytosol and NF-{kappa}B in the nucleus were adapted from a procedure described previously (69). Cell pellets were prepared as described above and lysed in boiling 1% SDS containing 1 mM sodium orthovanadate and 10 mM Tris, pH 7.4. Nuclear extracts were prepared as above. Fifty micrograms of cell lysate protein were electrophoresed on 12% SDS-polyacrylamide gels to measure I{kappa}B; 50 µg nuclear extract were electrophoresed on 8% SDS-polyacrylamide gels to measure NF-{kappa}B. Proteins were transferred to polyvinyldifluoride membranes, blocked for 1 h in 5% nonfat dry milk, and then incubated for 1 h with polyclonal antibodies against p65 or I{kappa}B (Rockland Immunochemicals, Gilbertsville, PA). The membrane was washed and developed using super signal chemiluminescence reagent (Pierce Chemical Co., Rockland, IL)

Other Assays and Statistical Significance
Recombinant proteins were produced using the pET system (Novagen, Madison, WI) as described previously (31, 33, 34). Protein concentration was determined using a BCA protein assays kit (Pierce Chemical Co.); crystalline BSA was the standard. For fluorescence-activated cell sorter (FACS) analysis, single cell suspensions were prepared and stained as described (14, 17, 70), using a class I-specific murine monoclonal antibody. Leu-4 was used as a background control.

All experiments were repeated at least three times with different batches of cells. Values are the mean ± SD. Significance between experimental values was determined by two-way ANOVA and was P < 0.05 or better when data from all experiments were considered.


    FOOTNOTES
 
Address requests for reprints to: Professor Fabrizio Monaco, Chair of Endocrinology, University "G. D’Annunzio", Palazzina Scuole di Specializzazione, Via dei Vestini, 66100 Chieti, Italy.

Received for publication July 6, 1999. Revision received January 5, 2000. Accepted for publication January 21, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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