Iodide Suppression of Major Histocompatibility Class I Gene Expression in Thyroid Cells Involves Enhancer A and the Transcription Factor NF-{kappa}B

Shin-Ichi Taniguchi1, Minho Shong2, Cesidio Giuliani3, Giorgio Napolitano3, Motoyasu Saji4, Valeria Montani3, Koichi Suzuki, Dinah S. Singer and Leonard D. Kohn

Cell Regulation Section (S.-I.T., M.S., C.G., G.N., M.S., V.M., K.S., L.D.K.) Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases and Experimental Immunology Branch (D.S.S.) National Cancer Institute National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
High concentrations of iodide can induce transient, clinical improvement in patients with autoimmune Graves’ disease. Previous work has related this iodide action to the autoregulatory effect of iodide on the growth and function of the thyroid; more recently, we additionally related this to the ability of iodide to suppress major histocompatibility (MHC) class I RNA levels and antigen expression on thyrocytes. In this report, we describe a transcriptional mechanism involved in iodide suppression of class I gene expression, which is potentially relevant to the autoregulatory action of iodide. Transfection experiments in FRTL-5 cells show that iodide decreases class I promoter activity and that this effect can be ascribed to the ability of iodide to modulate the formation of two specific protein/DNA complexes with enhancer A, -180 to -170 bp, of the class 1 5'-flanking region.1 Thus, iodide decreases the formation of Mod-1, an enhancer A complex involving the p50 subunit of NF-{kappa}B and a c-fos family member, fra-2, which was previously shown to be important in the suppression of class I levels by hydrocortisone. Unlike hydrocortisone, iodide also increases the formation of a complex with enhancer A, which we show, in antibody shift experiments, is a heterodimer of the p50 and p65 subunits of NF-{kappa}B. The changes in these complexes are not duplicated by chloride and are related to the action of iodide on class I RNA levels by the following observations. First, FRTL-5 thyroid cells with an aged phenotype coincidentally lose the ability of iodide to decrease MHC class I RNA levels and to induce changes in either complex. Second, the effect of iodide on class I RNA levels and on enhancer A complex formation with Mod-1 and the p50/p65 heterodimer is inhibited by agents that block the inositol phosphate, Ca++, phospholipase A2, arachidonate signal transduction pathway: acetylsalicylate, indomethacin, and 5,8,11,14-eicosatetraynoic acid. Interestingly, iodide can also decrease formation of the Mod-1 complex and increase formation of the complex with the p50/p65 subunits of NF-{kappa}B when the NF-{kappa}B enhancer sequence from the Ig {kappa} light chain, rather than enhancer A, is used as probe; and both actions mimic the action of a phorbol ester. This suggests that iodide may regulate complex formation with NF-{kappa}B regulatory elements on multiple genes associated with growth and function, providing a potential mechanism relating the autoregulatory action of iodide on thyroid cells and its action on class I gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Major histocompatibility complex (MHC) class I molecules are cell surface glycoproteins that play a pivotal role in immune responses and are important in the development of autoimmunity (1, 2, 3). Thus, there is overexpression of class I, as well as aberrant class II expression, in the pancreatic islet cells of type I diabetics and in thyrocytes from patients with autoimmune thyroid disease (ATD) (4, 5). {alpha}-Interferon, which increases class I but does not cause aberrant class II expression (6), can mimic autoimmune changes in class I levels in thyrocytes and other cells in culture (1, 2, 7) and can enhance or induce the appearance of thyroid autoantibodies and ATD when used to treat patients with hepatitis or cancer (8, 9, 10). Most importantly, class I deficient mice do not develop autoimmunity in several disease models, i.e. systemic lupus erythematosus (11), diabetes (12, 13), or autoimmune blepharitis (14); and methimazole, a drug used to treat patients with Graves’ disease, can suppress class I levels in rat FRTL-5 thyroid cells (7) and mimic the class I deficient state to prevent systemic lupus erythematosus and autoimmune blepharitis in those experimental models (14, 15).

High concentrations of iodide have been used in the past to treat patients with Graves’ disease; although only transiently effective, iodide is still used to prepare patients for surgery (16). The therapeutic action of high concentrations of iodide in ATD had been linked to its autoregulatory action on thyroid growth and function both in vivo (17) and in cultured FRTL-5 cells (18, 19, 20). In recent reports, we noted that high concentrations of iodide could also suppress class I levels in rat FRTL-5 and human thyroid cells (7, 21) and that as little as 4 days of high-iodide therapy could suppress class I levels in the thyroids of Graves’ patients being prepared for surgery (21). Coupled with the accumulating evidence for a role in class I overexpression in ATD, this suggested that the ability of iodide to suppress class I might be important in its therapeutic action in patients with Graves’ disease and that rat FRTL-5 cells might be a useful model with which to study this phenomenon.

FRTL-5 thyroid cells are a continuously cultured line whose growth and function, like other thyroid cells, depend not only on TSH/cAMP, but also on hydrocortisone, insulin, insulin-like growth factor-I (IGF-I), and other factors in serum (22, 23, 24). We recently showed that MHC class I expression in FRTL-5 cells was decreased by TSH/cAMP, hydrocortisone, insulin, IGF-I, and/or serum (7, 25 25A ), i.e. by all the hormones and factors necessary for thyroid cell growth and function. We hypothesized 1) that hormonal suppression of class I levels might be a normal mechanism to preserve self-tolerance in the face of hormone-induced increases in genes important for growth and function and 2) that common transcription factors might be involved in these coordinate actions (3, 7, 25 25A ). Understanding the mechanism of iodide suppression of class I gene expression in FRTL-5 cells might, therefore, enhance our understanding of the transcriptional mechanisms underlying self-tolerance and the autoregulatory actions of iodide on growth and function.

This report identifies a transcriptional mechanism by which iodide suppresses MHC class I gene expression in thyrocytes; it is the first description of a transcriptional action of iodide. We show that iodide regulates interactions between the ubiquitous transcription factor, NF-{kappa}B, and enhancer A of the MHC class I gene; and we link this action to the phosphoinositide-Ca++-arachidonate signal pathway, which is known to be involved in the growth and function of FRTL-5 thyroid cells (22). We show that the action of iodide is not restricted to enhancer A of the class I gene, but can similarly modulate complex formation with NF-{kappa}B elements on many genes implicated in the regulation of cell growth and function by ligands such as phorbol esters. A preliminary report of these data has been presented (25a).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of Iodide on Class I RNA Levels in Fresh vs. Aged Phenotype FRTL-5 Thyroid Cells
In a previous report (7), we showed that iodide could decrease class I RNA levels and that this effect was not mediated by cAMP. The effect of 1 mM iodide on class I RNA levels is evident within 3 h, significant within 6 h, and near maximal by 12 h in FRTL-5 cells with a low passage number (Fig. 1BGo). This rapid action is consistent with the rapid effects of high concentrations of iodide in clinical practice (17) and on growth or DNA synthesis in FRTL-5 cells (18, 19, 20). The effect of a single addition of iodide begins to wane after 24 h and is nonexistent 96 h after challenge (Fig. 1BGo); however, the decrease will persist over a 96-h period if fresh iodide is added at 24, 48, and 72 h after the start of the experiment (Table 1Go). Interestingly, the effect of iodide is lost in FRTL-5 thyroid cells with an aged phenotype, i.e. in cells with passage numbers > 30 (Fig. 1Go, A and B).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 1. Effect of Iodide as a Function of Time on MHC Class I RNA Levels in Rat FRTL-5 Thyroid Cells

After growth to near confluency, FRTL-5 cells, with a low passage number (<20) or with a high passage number (>30) were maintained for 6 days in 5H medium with no TSH plus 5% calf serum before 1 mM iodide was added. Cells with a high passage number have an aged phenotype characterized by high thymidine incorporation into DNA in the absence of TSH and an attenuated response to TSH over this basal value (26). At the noted times, RNA was isolated and subjected to Northern analysis using, sequentially, probes for MHC class I and ß-actin. In panel A, a representative Northern analysis from one experiment is presented, which contained 20 µg total RNA/lane (based on optical density). In panel B the ratio of class I RNA to ß-actin RNA levels was calculated after quantitative densitometry. Data are the mean ± SE from four independent experiments. Similar results were obtained using cells maintained in TSH (6H medium) plus 5% serum.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Effect of 1 mM Iodide on Class I RNA Levels When Added to the Medium Every 24 h as Opposed to a Single Addition of Iodide at the Start of the Experiment

 
The loss of iodide suppression in cells with an aged phenotype was surprising and initially difficult to understand. However, it should be recalled that FRTL-5 thyroid cells with an aged phenotype are characterized by a very high basal thymidine incorporation into DNA, at least 10- to 20-fold higher than freshly passaged cells (26). This is associated with an altered inositol phosphate-Ca++-arachidonate signal pathway and an increase in cyclooxygenase and prostaglandin-E2 isomerase-like activities resulting from an increased responsiveness to insulin/IGF-I/serum (26, 27). As will be noted below, iodide signaling of the changes in RNA levels and class I transcription involves the inositol phosphate-Ca++-arachidonate-cyclooxygenase signal pathway; thus, the loss of the iodide response in aged cells is consistent with the alteration in the iodide-induced signal that generates the effect. In this regard, it should also be noted that class I RNA levels are significantly higher, 2- to 3-fold, in cells with higher passage numbers and an aged phenotype, by comparison to cells with a low passage number (Fig. 1Go). This may be relevant to the fact that autoimmune thyroid disease tends to occur with aging, even in families with a history of thyroid autoimmunity.

The ability of iodide to suppress class I levels was dependent on the concentration of iodide and was evident at higher iodide concentrations associated with iodide autoregulation in these cells (17, 18, 19, 20); inhibition was only 21 ± 6% of control values at 0.1 mM iodide, but 51 ± 8% at 0.5 mM, 66 ± 4% at 1 mM, and 78–80 ± 7% at 5 or 10 mM. Data are the mean ± SE from four independent experiments and represent a significant decrease in RNA (P < 0.05 or better). Suppression was not duplicated by chloride ions, which are present at >100 mM concentrations in the controls without iodide. The effect was evident in FRTL-5 cells maintained with (data not shown) or without (Fig. 1Go) TSH in the medium.

The Effect of Iodide on Class I RNA Levels Reflects an Action Involving Enhancer A of the Class-I Promoter
Iodide Regulates Promoter Activity via Enhancer A

At a concentration of 1 mM, which is effective in decreasing endogenous class I RNA levels (Fig. 1Go), iodide decreased the activity of an exogenous class I promoter containing 1100 bp of class I 5'-flanking region that had been ligated to a chloramphenicol acetyltransferase (CAT) reporter gene, p(-1100)CAT, and that had been transfected into FRTL-5 thyroid cells (Fig. 2Go). Using 5'-deletion mutants, the iodide effect could be localized to between -203 and -127 bp of the start of transcription (Fig. 2Go). This region contains two major regulatory elements, enhancer A, -180 to -170 bp, and the interferon response element, -161 to -150 bp (Fig. 2Go, bottom).



View larger version (42K):
[in this window]
[in a new window]
 
Figure 2. Effect of 1 or 10 mM Iodide on the Promoter Activity of FRTL-5 Thyroid Cells Transfected with Chimeric Swine PD1 MHC Promoter-CAT Constructs with Different 5'-Extensions and with -203-bp CAT Chimeras of the Class I Promoter with [p(203 M)] or without [p(-203)] a Mutation of the Enhancer A Sequence

A diagrammatic representation of the swine class I p(-203)CAT chimera is at the bottom of the figure. The TATA and CCAT boxes are noted, as are the CRE-like sequence, -107 to -100 bp, as well as the interferon response element (IRE) and enhancer A, between -203 and -127 bp. The wild-type sequence of enhancer A in p(-203), as well as a mutant sequence in p(-203 M), which results in the loss of hydrocortisone-induced suppressive activity on MHC class I (28), is presented at the bottom of Fig. 4Go (Mutant 2) and in the text. All numbers are relative to the start of transcription. FRTL-5 cells were transfected with the noted plasmids using a DEAE-dextran procedure (Materials and Methods). Twenty-four hours after transfection, 10 mM sodium chloride was added to the medium of control cells and 1 or 10 mM sodium iodide was added to the experimental groups. CAT activity was measured 24 h later. Efficiency of transfection was determined by cotransfection with 5 µg pRSVLuc, kindly provided by Dr. S. Subramani (University of California, La Jolla). CAT values, mean ± SE of three experiments, are normalized to luciferase activity and protein using the Promega assay system and a Moonlight 2010 luminometer. Values are expressed as the percent of the control value for the noted plasmid in the absence of iodide; control values for the p(-203)CAT and p(-203 M)CAT constructs were approximately the same. One star denotes a significant iodide-induced decrease in CAT activity (P < 0.05); two stars denote a significant decrease at P < 0.01. CAT activity is measured as described in Materials and Methods. Similar results were obtained using FRTL-5 cells stably transfected with chimeric swine PD1 MHC promoter-CAT constructs (data not shown).

 
The effect was dependent on the concentration of iodide (Fig. 2Go) and was lost in a construct, p(-203 M)CAT, which has a mutation (termed Mutant 2) of enhancer A, from 5'-GGGGAGTCCCC-3' (-180 to -170 bp) to 5'-GCCGAGTCAAG-3' (Fig. 2Go and Table 2Go). The same result, a loss of iodide suppression, was also seen using another p(-203)CAT enhancer A mutation (termed Mutant 1) to 5'-GCGGAGTCAAG-3' (Table 2Go). Enhancer A is the element we linked to the ability of hydrocortisone to suppress MHC class I gene expression; the same mutations eliminate the ability of hydrocortisone to decrease MHC class I gene expression (28).


View this table:
[in this window]
[in a new window]
 
Table 2. Effect of 1 mM Iodide on the Promoter Activity of FRTL-5 Thyroid Cells Transiently Transfected with p(-203) CAT Chimeras of the Class I Promoter Containing Mutations of the Enhancer A (EnhA) or Interferon Response Element (IRE)

 
A mutation of the interferon response element, from 5'-AGTTTCACTTCTCC-3' (-161 to -148 bp) to 5'-AGTGGTCCTTCTCC-3' or to 5'-AGTTTCCTTTCTCC-3', Mutant 1 and 2, respectively, which can each decrease the interferon-induced increase in class I levels (data not shown), did not alter the ability of iodide to decrease p(-203)CAT activity (Table 2Go). This suggested that the iodide effect on class I gene expression, like hydrocortisone, involved the enhancer A element.

Iodide Regulates the Formation of Two Protein/DNA Complexes with Enhancer A: Mod-1 and a p50/p65 Heterodimer
Hydrocortisone decreases formation of a protein complex with enhancer A, -180 to -170 bp, in the class I 5'-flanking region, termed Mod-1, which involves the p50 subunit of NF-{kappa}B and a c-fos family member, fra-2 (28). Using the 74-bp region between -203 and -130 bp as a labeled probe, we could show that iodide treatment of FRTL-5 cells for as little as 1.5 h resulted in extracts with a reduced ability to generate a protein/DNA complex located toward the top of the electrophoretic mobility shift analysis (EMSA) gel (Fig. 3Go, lane 3 vs. 2). This complex was identified as Mod-1 by the ability of antibodies to the p50 subunit of NF-{kappa}B (Fig. 3Go, lane 4) and to fra-2 (Fig. 3Go, lane 9) to inhibit its formation and/or supershift the complex decreased by iodide. Antibodies to the p65 subunit of NF-{kappa}B, p52, c-fos, fra-1, or c-jun had no effect on the Mod-1 complex (Fig. 3Go, lanes 5 to 8 and 10, respectively).



View larger version (62K):
[in this window]
[in a new window]
 
Figure 3. Effect of Iodide on the Formation of the Mod-1 Protein/DNA Complex between FRTL-5 Thyroid Cell Extracts and a 74-bp Fragment of the MHC Class I Promoter between -203 and -130 bp

FRTL-5 thyroid cells with a low passage number (<20) were grown to near confluency in 6H medium containing 5% calf serum and then maintained for 6 days in 5H medium with 5% calf serum (but no TSH). Cells were fed fresh medium with or without 1 mM iodide and extracts prepared after 1.5 h. Extracts were incubated with a radiolabeled 74-bp fragment of the MHC class I promoter between -203 and -130 bp, which is diagrammatically depicted at the bottom of the figure. Incubations were in a low salt buffer without detergents as described in Materials and Methods. EMSAs were used to identify protein DNA complexes. Lane 1 (None) 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 1 mM iodide. Lanes 4–10 present the effect of rabbit polyclonal antibodies (Santa Cruz) to the noted transcription factors on the interaction between the iodide-treated extract and the radiolabeled 74-bp fragment. The same results were obtained using control extracts from cells without exposure to iodide, as evidenced in Fig. 4Go, where radiolabeled enhancer A is the probe, i.e. antibody specificity was the same in extracts from cells treated with or without iodide. Mod-1 is noted as the protein/DNA complex whose formation is regulated by iodide and whose formation is inhibited and/or supershifted by anti-p50 (lane 4) and anti-Fra2 (lane 9).

 
The ability of iodide to decrease the Mod-1 complex with enhancer A was also evidenced in experiments using a shorter radiolabeled oligonucleotide, -187 to -164 bp, which encompasses enhancer A, -180 to -170 bp (Fig. 4Go). In this experiment, iodide treatment of FRTL-5 cells decreased formation of a major complex formed with these extracts (Fig. 4AGo, lane 2 vs. 1). This is the Mod-1 complex, as evidenced by the ability of antibodies to the p50 subunit of NF-{kappa}B (Fig. 4BGo, lane 3) or fra-2 (Fig. 4BGo, lane 7) to prevent its formation and/or supershift the complex, but not antibodies to the p65 subunit of NF-{kappa}B, p52, c-fos, fra-1, or c-jun (Fig. 4BGo, lanes 4 to 6 and 8 and 9, respectively). These studies showed the effect of the two antibodies on the complexes in extracts from cells before iodide treatment, whereas the data in Fig. 3Go showed the effect of the antibodies on the complexes in cells treated with iodide. The data thus indicate that the antibody specificities are not affected by iodide treatment of the cells. More importantly, the data in both figures show that there is no complex in this region that is not Mod-1 and that the Mod-1 complex with enhancer A is decreased by iodide treatment of the cells. Consistent with this, formation of the complex was inhibited by the presence of a 100-fold excess of unlabeled enhancer A oligonucleotide in the binding mixture (Fig. 4BGo, lane 11 vs. 10), but not by a 100-fold excess of oligonucleotides with the mutations in the enhancer A site (Fig. 4BGo, lanes 12 and 13) that result in a loss of the ability of iodide to suppress class I promoter activity (Fig. 4Go and Table 2Go).



View larger version (24K):
[in this window]
[in a new window]
 
Figure 4. Formation of the Mod-1 Protein/DNA Complex between FRTL-5 Thyroid Cell Extracts and an Oligonucleotide with the Sequence of Enhancer A of the MHC Class I Promoter between -203 and -130 bp

The sequences of the enhancer A oligo and its two mutant derivatives are presented at the bottom of the figure. These are the same enhancer A mutations that cause the CAT chimeric constructs of p(-203) to lose iodide suppression of class I promoter activity (Fig. 2Go and Table 2Go). After growth to near confluency, FRTL-5 thyroid cells with a low passage number (<20) were maintained for 6 days in 5H medium plus 5% calf serum (no TSH) and treated with or without 1 mM iodide for 1.5 h. Extracts were prepared and incubated with the radiolabeled oligonucleotide having the enhancer A sequence of the MHC class I promoter. Incubations were in a low-salt buffer without detergents, as described in Materials and Methods and as used in Fig. 3Go. EMSAs were used to identify protein/DNA complexes. In panel A, lanes 1 and 2 are, respectively, incubations of radioactive probe and extracts of control cells or cells treated with 1 mM iodide. In panel B, extracts are from control cells not treated with iodide. Lane 1 contains the probe alone, lane 2 contains probe plus extract, and lanes 3–10 contain the probe plus extract incubated in the presence of rabbit polyclonal antibodies to the noted transcription factors. Lanes 11–13 show the effect of unlabeled oligonucleotide competitors on complex formation, either the wild-type enhancer A oligonucleotide or oligos with mutant enhancer A sequences noted at the bottom of the figure, each in a 100-fold excess. The same results were obtained using extracts from iodide-treated cells, as evidenced in Fig. 3Go. The protein/DNA complex whose formation is regulated by iodide is identified as Mod-1 based on inhibition and/or supershifts in the presence of anti-p50 (lane 3) and anti-Fra2 (lane 7).

 
Iodide does more, however, than decrease the Mod-1 complex; it also increases formation of a complex with enhancer A involving a heterodimer of the p50 and p65 subunits of NF-{kappa}B. This is measurable using conditions, detergents and high salts, that favor formation of this complex, but appear to restrict formation of the Mod-1 complex. Thus, iodide increases formation of a complex with the radiolabeled enhancer A oligonucleotide (Fig. 5AGo, lane 3 vs. 2) in the presence of detergent and high salts. The complex is specific, since its formation is prevented by unlabeled enhancer A (Fig. 5AGo, lane 4) but not by either of its mutants (Fig. 5AGo, lanes 5 and 6), whose sequences are noted on the bottom of Fig. 4Go and which cause a loss of iodide-induced suppression of class I promoter activity (Fig. 2Go and Table 2Go). The complex is decreased and supershifted by an antibody to the p50 subunit of NF-{kappa}B and decreased by an antibody to the p65 subunit of NF-{kappa}B (Fig. 5AGo, lanes 9 and 10, respectively), but unaffected by antibodies to c-rel, fra-1, fra-2, c-fos, or c-jun (Fig. 5AGo, lanes 11–15).



View larger version (60K):
[in this window]
[in a new window]
 
Figure 5. Effect of Iodide on the Formation of Protein/DNA Complexes between FRTL-5 Thyroid Cell Extracts and an Oligonucleotide with the Sequence of Enhancer A of the MHC Class I Promoter, but Measured under High-Salt Conditions in the Presence of Detergent

FRTL-5 thyroid cells with a low passage number (<20) were grown to near confluency and maintained for 6 days in 5H medium (no TSH) plus 5% calf serum. At that point, in cells were incubated with fresh medium with or without 1 mM iodide for 1.5 h (panel A). Extracts were prepared and incubated with the radiolabeled oligonucleotide with the enhancer A sequence of the MHC class I promoter; incubations were in a high-salt buffer with detergent as described in Materials and Methods, rather than the low-salt buffer without detergent used in Figs. 3Go and 4Go. EMSAs were used to identify protein DNA complexes. 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 1 mM iodide. Lanes 4–6 represent incubations with extracts from cells treated with iodide plus unlabeled oligonucleotide competitors with the wild-type or mutant sequences noted at the bottom of Fig. 4Go, each in a 100-fold excess. Lane 7 contains an incubation wherein extracts from cells treated with iodide were exposed to the enhancer A probe in the presence of a 100-fold excess of unlabeled oligonucleotide with the sequence of the NF-{kappa}B binding element from the enhancer sequence of the Ig {kappa} light chain, the sequence of which is noted at the top of Fig. 8Go. Lanes 8–15 depict the effect of serum from a normal rabbit or rabbit polyclonal antibodies to the noted transcription factors on the interaction between the iodide-treated extract and the radiolabeled probe. Arrows denote the location of the iodide-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 on the antisera data and its mobility on a gel relative to complexes formed by different concentrations of authentic p50 protein. In panel B, cells were exposed to 1 mM iodide for the noted times (lanes 3–10) or to the noted iodide concentrations (lanes 12–14) for 1.5 h.

 
The following additional points concerning the effect of iodide on these complexes should be noted. First, the ability of iodide to decrease Mod-1 (Figs. 3Go and 4Go) or increase formation of the p50/p65 heterodimer complex (Fig. 5AGo) was rapid, preceding the change in RNA (Fig. 1Go), and was consistent with the changes in class I RNA with time after a single exposure to iodide (Fig. 1Go). Thus it was evident within 60 min of iodide (1 mM) challenge, was maximal at 2 h, decreased by 3–6 h, and, although still evident at 24 h, as illustrated for p50/p65 heterodimer formation (Fig. 5BGo, lanes 3–10) after a single exposure to iodide, was beginning to wane. This is very different from the action of hydrocortisone in two respects. Hydrocortisone does not increase p50/p65 complex formation with enhancer A when evaluated under comparable conditions; and the ability of hydrocortisone to decrease Mod-1 requires 24–48 h of FRTL-5 cell treatment, comparable to its effect as a function of time on RNA levels and promoter activity (28). The ability of iodide to both increase p50/p65 complex formation (Fig. 5AGo) or decrease Mod-1 (Figs. 3Go and 4Go) was also evident only at higher iodide concentrations, 0.1 to 10 mM, like the class I RNA decrease, as illustrated for p50/p65 heterodimer formation (Fig. 5BGo, lanes 12–14); it was not evident at 0.01 mM or lower concentrations of iodide (data not shown).

Second, the effect of iodide on formation of the p50/p65 heterodimer complex is very specific and does not reflect a generalized action on NF-{kappa}B subunits. Thus, a second, non-iodide-induced, faster moving complex with enhancer A is inhibited only by anti-p50 (Fig. 5AGo, lane 9). This property, plus its migration on gels with respect to different concentrations of authentic p50 protein (data not shown), is consistent with its identification as a p50 homodimer. Iodide regulates, therefore, formation of only the p50/p65 heterodimer complex with enhancer A and not formation of the p50 homodimer complex.

Last, enhancer A has a core sequence, GGGGA, which is common to NF-{kappa}B binding elements from other genes, e.g. the enhancer sequence from the Ig {kappa} light chain. A 22 mer oligonucleotide containing the enhancer sequence from the Ig {kappa} light chain can prevent formation of both the iodide-induced p50/p65 heterodimer and p50 homodimer (Fig. 5AGo, lane 7), but not the lower complexes. We previously showed that the same 22 mer oligonucleotide containing the enhancer sequence from the Ig {kappa} light chain could prevent formation of Mod-1 (28). With the exception of the core GGGGA sequence, the sequence of the oligonucleotide containing enhancer A of class I, 5'-CGGTGGTGGGGAGTCCCCGTGTCC-3', differs from 5'-AGTTGAGGGGACTTTCCCAGGC-3', the Ig {kappa} light chain enhancer, in that it has 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 iodide-induced p50/p65 heterodimer complex, just as it is for the iodide-decreased Mod-1 complex, as previously demonstrated (28). The import of this observation will be further noted in experiments to be described and discussed below.

The Effect of Iodide on Class I Gene Expression Is Linked to Its Effect on Complex Formation; Both Effects Are Mediated by an Action Involving the Phosphoinositide-Ca++-Arachidonic Acid-Cyclooxygenase Signal Transduction Pathway

The ability of iodide to decrease Mod-1 and increase the p50/p65 heterodimer complex was functionally linked to the action of iodide on class I RNA levels by the following experiments.

First, we had shown that iodide did not decrease class I RNA levels in FRTL-5 cells with an aged phenotype (Fig. 1Go). Iodide also did not decrease the Mod-1 complex in FRTL-5 cells with an aged phenotype (Fig. 6AGo, lanes 3 vs. 2 or 5 vs. 4); nor did iodide increase the formation of the p50/p65 heterodimer complex (Fig. 6BGo, lanes 3 vs. 2 or 5 vs. 4). This was not true for a phorbol ester, o-tetradecanoyl phorbol 13-acetate (TPA), which can increase formation of the p50/p65 heterodimer and the p50 homodimer (Fig. 6BGo, lane 6 vs. 2) in aged cells, unlike its vehicle, dimethylsulfoxide (Fig. 6BGo, lane 7 vs. 2), and served as a positive control. These data indicate that both the iodide-induced decrease in the Mod-1 complex with enhancer A and the iodide-induced increase in the p50/p65 heterodimer complex are lost concurrently with the loss in iodide-induced decreases in class I RNA levels. The effect of the phorbol ester on class I RNA levels is similar to that of iodide, as preliminarily reported (28a); this will be presented separately (S.-I. Taniquchi, M. Shong, V. Montani, C. Giuliani, M. Saji, D. S. Singer, and L. D. Kohn, manuscript in preparation).



View larger version (33K):
[in this window]
[in a new window]
 
Figure 6. Effect of Iodide Treatment of Aged FRTL-5 Thyroid Cells on the Formation of Protein/DNA Complexes between Extracts and the Enhancer A Oligonucleotide Probe, Measured in Low Salts without Detergent (A) or under High-Salt Conditions in the Presence of Detergent (B)

FRTL-5 thyroid cells with a high passage number (>30) and an aged phenotype were grown to near confluency in 6H medium and then were maintained for 6 days in 5H medium with no TSH plus 5% calf serum. Cells were, at that point, treated with 1 mM iodide for 1.5 or 24 h or with TPA or the dimethyl sulfoxide-containing buffer present with the TPA for 1.5 h. Extracts were prepared and incubated with a radiolabeled oligonucleotide having the enhancer A sequence of the MHC class I promoter, either in a low-salt buffer without detergents exactly as in Figs. 3Go and 4Go (panel A) or in the high-salt buffer plus detergent exactly as in Fig. 5Go (panel B). EMSAs were used to identify protein DNA complexes. The Mod-1, p50/p65 heterodimer, and p50 homodimer complexes are defined by antisera inhibition as in Figs. 3–5GoGoGo (data not shown).

 
Second, we showed that the reduction of RNA levels and modulation of complex formation by iodide were simultaneously inhibited by agents that inhibit the phosphoinositide-Ca++-arachidonic acid-cyclooxygenase signal transduction pathway in FRTL-5 cells with a low passage number. We performed this experiment because FRTL-5 thyroid cells with an aged phenotype are characterized by an altered inositol phosphate-Ca++-arachidonate-signal pathway with increased cyclooxygenase and prostaglandin-E2 isomerase-like activities (26, 27) and because the autoregulatory action of iodide on thyroid function had been linked to the inositol phosphate-Ca++-arachidonate signal pathway in studies of H2O2 production in dog thyroid cells in culture (29). We therefore evaluated the effect of inhibitors of this pathway on the ability of iodide to simultaneously modulate complex formation and class I RNA levels.

As illustrated in assays measuring p50/p65 heterodimer and p50 homodimer complex formation with enhancer A, the ability of iodide to increase complex formation (Fig. 7AGo, lane 7 vs. 6; Fig. 7BGo, lane 5 vs. 2) in extracts from cells with low passage numbers was inhibited by the phospholipase A2 (PLA2) inhibitor, acetyl salicylate (ASA); by the cyclooxygenase inhibitor, indomethacin (Fig. 7AGo, lanes 8 and 9, respectively, vs. lane 7); and by 5,8,11,14-eicosatetraynoic acid (ETYA) (Fig. 7BGo, lane 6 vs. 5), which inhibits cyclooxygenase, lipoxygenase, epoxygenase, and PLA2 activities. The ability of these agents to inhibit iodide-induced p50/p65 heterodimer complex formation appeared to be specific, since none of the agents blocked the ability of a maximally effective concentration of TPA, 50 nM, to increase p50/p65 heterodimer or p50 homodimer complex formation (Fig. 7AGo, lanes 4 and 5 vs. lane 3; Fig. 7BGo, lane 4 vs. 3). Additionally, none of the agents, ASA, indomethacin, or ETYA, inhibited basal complex formation in control extracts (Fig. 7CGo). Moreover, the ability of ASA, indomethacin, and ETYA to inhibit iodide-increased p50/p65 heterodimer complex formation with enhancer A was coincident with their abilities to inhibit the ability of iodide to decrease class I RNA levels and decrease Mod-1 complex formation and was again specific with respect to the action of TPA (Table 3Go).



View larger version (41K):
[in this window]
[in a new window]
 
Figure 7. Effect of Agents Inhibiting the Phosphoinositide-Ca++-Arachidonic Acid Signal Transduction System on the Ability of FRTL-5 Cells Treated with 1 mM Iodide or 50 nM TPA to Increase p50/p65 Heterodimer Complex Formation

FRTL-5 cells with a low passage number (<20) were grown to near confluency and maintained for 6 days in 5H medium with no TSH plus 5% calf serum before 1 mM iodide or 50 nM TPA was added for 1.5 h. Complex formation with the radiolabeled enhancer A oligonucleotide was measured in the high-salt/detergent buffer system as in Fig. 5Go. In panel A, the effect of 1 mM ASA and 0.1 mM indomethacin were tested on TPA- or iodide-increased complexes; in panel B the effect of 1 mM ETYA was measured on TPA- or iodide-increased complexes; in panel C the effect of 1 mM ASA, 0.1 mM indomethacin, and 1 mM ETYA was measured on complexes from control cells. Inhibitors were added at the same time as the iodide or TPA. In panels A and B, lane 1 contains the probe alone and lane 2 contains the probe with an extract from control cells not treated with iodide or TPA. A diagrammatic representation noting the locus of action of the inhibitors is noted in panel D; the putative sites wherein iodide might activate the phosphoinositide-Ca++-arachidonic acid signal transduction pathway to induce complex formation are noted as is the site of action of TPA.

 

View this table:
[in this window]
[in a new window]
 
Table 3. Inhibitory Effect of Drugs on the Ability of 1 mM Iodide or 50 nM TPA to Decrease Mod-1 Complex Formation with Enhancer A, Increase p50/p65 Heterodimer Complex Formation with Enhancer A, or Decrease MHC Class I RNA Levels

 
These data (Figs. 6Go and 7Go and Table 3Go) support the following conclusions. The iodide-induced changes in the Mod-1 and p50/p65 complexes with enhancer A are involved in class I suppression, since they correlate with iodide-induced changes in RNA levels. Iodide acts to alter complex formation and decrease class I levels by activating the phospholipase A2-arachidonic acid signal transduction system (Fig. 7DGo). Additionally, iodide modulation of complex formation differs mechanistically from that of a phorbol ester, since TPA activity is not inhibited by ASA, indomethacin, or ETYA (Fig. 7DGo). Phorbol esters appear to act via protein kinase C; as noted above, a separate report will address the effect of phorbol esters on class I activity and has been presented in preliminary fashion (28a).

Two points should be noted before continuing. The persistent ability of TPA to alter complex formation in FRTL-5 cells with an aged phenotype (Fig. 6Go), which have an altered inositol phosphate-Ca++-arachidonate-cyclooxygenase signal pathway (26), is consistent with the insensitivity of TPA action to inhibitors of this pathway (Fig. 7Go). Additionally, and also consistent with the data in Figs. 6Go and 7Go, the PLA2 activator, mellitin (5 µg/ml), as well as the arachidonic acid metabolites prostaglandin E2 (10 µM) and prostaglandin F2{alpha} (10 µM), increased p50/p65 heterodimer complex formation in FRTL-5 cells with a low passage number but not in cells with a high passage number (data not shown).

Iodide Regulates Complex Formation with Elements from Other Genes Interacting with the Subunits of NF-{kappa}B
Enhancer A of the class I promoter has a core sequence that is present in NF-{kappa}B binding elements from other genes, e.g. the enhancer sequence from the Ig {kappa} light chain (Fig. 8Go, top). As noted above (Fig. 5AGo), an oligonucleotide containing the core sequence of the NF-{kappa}B binding element of the enhancer sequence from the Ig {kappa} light chain will prevent formation of the iodide-induced p50/p65 complex with the enhancer A oligonucleotide from the class I gene. We therefore evaluated the effect of iodide on complex formation with the NF-{kappa}B binding element to determine whether iodide might, as a first approximation, be capable of regulating genes other than MHC class I in thyrocytes.



View larger version (51K):
[in this window]
[in a new window]
 
Figure 8. Effect of Iodide Treatment of Recently Passaged FRTL-5 Thyroid Cells on the Formation of Protein/DNA Complexes between Extracts and the NF-{kappa}B Enhancer Element from the Ig {kappa} Light Chain, Measured in Low Salts without Detergent (A) or under High-Salt Conditions in the Presence of Detergent (B-D)

In panels A and B, FRTL-5 thyroid cells with a low passage number (<20) were grown to near confluency and maintained for 6 days in 5H medium with no TSH plus 5% calf serum before being exposed to fresh medium with or without 1 mM sodium chloride (Control) or sodium iodide for 1.5 h. Extracts were prepared and incubated with the radiolabeled oligonucleotide having the sequence of the NF-{kappa}B enhancer element from the Ig {kappa} light chain, which is depicted at the top of the figure. Incubations were either in a low- salt buffer without detergents exactly as in Figs. 3Go and 4Go (panel A) or in the high-salt buffer plus detergent exactly as in Fig. 5Go (panels B–D). In panel C, cells were treated with 1 mM sodium iodide for different time periods before extracts were evaluated to measure p50/p65 complex formation; in panel D cells were treated with the noted concentrations of iodide, and extracts were obtained 1.5 h later.

 
When radiolabeled, the NF-{kappa}B binding element from the Ig {kappa} light chain will form a protein/DNA complex at the top of the gel under low salt conditions without detergent, which is decreased in extracts from iodide-treated cells (Fig. 8AGo, lane 3 vs. 2). This is Mod-1 as evidenced by the ability of anti-p50 and anti-fra-2 to inhibit its formation and supershift the complex (Fig. 8AGo, lanes 5 and 6, respectively), but not antibodies to p65, fra-1, c-fos, or c-jun (data not shown).

Additionally, extracts from iodide-treated cells will form a p50/p65 heterodimer complex with the NF-{kappa}B binding element when incubated in high salts plus detergent (Fig. 8BGo, lane 3 vs. 2). The identification of the p50/p65 complex, whose formation is induced by iodide, is evidenced by its reactivity with anti-p50 (Fig. 8BGo, lane 4) and anti-p65 (Fig. 8BGo, lane 5) but not anti-p52, anti-c-rel, or anti-fra-2 (Fig. 8BGo, lanes 6–8). The effect of iodide treatment of the cells is rapid, as evidenced by induction of the p50/p65 heterodimer complex (Fig. 8CGo), and depends on high iodide concentrations (Fig. 8DGo) as is the case for the formation of this complex with enhancer A (Fig. 5Go, A and B), although slight differences cannot be excluded.

The effect of iodide on both Mod-1 and the p50/p65 heterodimer complex formed by the NF-{kappa}B binding element from the Ig {kappa} light chain is duplicated by a phorbol ester (see Figs. 3–7GoGoGoGoGo). Further, and again unlike iodide, the phorbol ester increases p50 homodimer formation as illustrated in Fig. 7Go, A and B. These complexes were again identified using the antibodies.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Increased MHC class I expression on thyrocytes is evident in ATD (4, 21) and has been suggested to be an important causal factor in the development or perpetuation of disease expression (3). The hypothesis has been offered that the elevations result in abnormal presentation of thyroid antigens to immune cells, thereby breaking tolerance, and that this elicits the clonal expansion of autoreactive T cells associated with disease expression (3). The possibility that the thyroid cells can become antigen-presenting cells is supported by the observations that human thyroid cells express components of the complex machinery involved in antigen presentation, such as intercellular adhesion molecule-1 (ICAM-1) and invariant chain (30), that there is aberrant class II expression in ATD (4, 31), and that interferon can induce aberrant class II expression, as well as increase class I expression in human and FRTL-5 thyrocytes in culture (32, 33, 34, 35).

Iodide therapy in Graves’ disease is a well recognized treatment whose effectiveness is associated with the Wolf-Chaikoff effect (17, 36, 37), wherein, for example, high concentrations of iodide suppress thyroid hormone secretion. Iodide therapy is additionally associated with the autoregulatory action of iodide that affects the growth of the thyroid (16, 17, 18, 19, 20). The mechanisms underlying both the iodide-immunosuppressive action in Graves’ patients and iodide autoregulation of growth and function remain largely unclear (16, 17, 37). In recent studies (7, 21), we showed that iodide at high concentrations, the same as those used to study its autoregulatory action (18, 19, 20, 38), could decrease MHC class I surface expression and/or RNA levels in human and rat FRTL-5 thyrocytes. Moreover, we showed that preoperative iodide treatment of Graves’ patients suppressed MHC class I RNA levels in the Graves’ thyroid (21). In this report we describe a transcriptional action of iodide that can account for its immunosuppressive action on MHC class I gene expression in Graves’ and that provides a potential explanation for its complex autoregulatory action on growth and function by its relationship to the transcriptional mechanism of action of phorbol esters. Thus, this is the first recognition that iodide regulation of class I gene expression and thyroid growth or function, like phorbol esters, involves the NF-{kappa}B system and the complex array of its subunit interactions with the jun/fos or rel transcription factors.

We show in this report that high concentrations of iodide, like hydrocortisone (28), can decrease the formation of a protein/DNA complex, termed Mod-1, with enhancer A of the class I promoter. Unlike the case of hydrocortisone (28), however, we show that iodide can simultaneously increase the formation of a new and different complex with enhancer A, which, like Mod-1, involves the subunits of the NF-{kappa}B-regulatory transcription factor. Mod-1 is a complex (28) involving the p50 subunit of NF-{kappa}B and a member of the a fos family member, fra-2 (39); the new complex is a heterodimer of the p50 and p65 subunits of NF-{kappa}B. We show that a critical recognition element for the binding of both complexes is the GGGGA sequence within enhancer A and that the iodide action on the formation of each complex and on enhancer A-dependent class I promoter activity correlates with its ability to suppress MHC class I RNA levels. Thus, we show that aged cells concurrently lose these activities and that inhibitors of the ability of iodide to modulate the arachidonate signal transduction system also concurrently inhibit them.

Hydrocortisone suppresses class I by decreasing the formation of the Mod-1 complex with enhancer A, -180 to -170 bp in its 5'-flanking region (28). In contrast, interferon increases the binding of Mod-1 to enhancer A, in association with its ability to increase class I expression (28). Enhancer A is, therefore, an important element involved in the interferon response, in addition to the interferon response element (28, 40, 41); suppression of the Mod-1 interaction with enhancer A by anti-immune agents, such as hydrocortisone, is one means of counteracting the action of interferon and, by extension, the autoimmune response. The basis for this appears to be the fact that the p50 component of Mod-1 has a footprint that overlaps the enhancer A and interferon response elements (28). These data are consistent with separate data which showed that down-regulation of the p50 subunit decreases MHC class I expression (42).

We suggest, therefore, that iodide, like hydrocortisone (28), decreases the Mod-1 complex with enhancer A, thereby decreasing MHC class I promoter activity and counterbalancing the action of cytokines such as interferon that increase complex formation and class I expression (Fig. 9Go). Unlike hydrocortisone, iodide also increases the formation of a protein/DNA complex between enhancer A and a p50/p65 heterodimer of NF-{kappa}B subunits (Fig. 9Go), and this action, as well as a rapid decrease in Mod-1, is duplicated by phorbol esters, which also can suppress class I activity and RNA levels in FRTL-5 thyroid cells, as preliminarily reported (28a).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 9. Schematic Model of the Interaction of Mod-1 and the p50/p65 Heterodimer Complex with Enhancer A of the MHC Class I Promoter

Whereas interferon increases Mod-1 complex formation with enhancer A, iodide as well as hydrocortisone decreases Mod-1 complex formation. Iodide, unlike hydrocortisone, increases the ability of enhancer A to bind a p50/p65 heterodimer form of NF-{kappa}B.

 
It is unclear, at this time, what functional role might be played by the increase in p50/p65 heterodimer complex formation or what functional interplay might exist between it and Mod-1. For example, does the binding of the p50/p65 heterodimer to enhancer A merely fill the void when Mod-1 is decreased or, more likely we believe, is its formation the impetus for the more rapid decrease in Mod-1 induced by iodide or phorbol esters than by hydrocortisone (Fig. 9Go)? Does it play a more important role in the activation of other genes with the ability to bind the p50/p65 heterodimer of NF-{kappa}B, as is the case for phorbol esters?

NF-{kappa}B is an inducible eukaryotic transcription factor of the rel family. It exists in the cytoplasm of most cells in an inactive form, where it is bound to I{kappa}B (43, 44, 45). NF-{kappa}B is activated in response to a number of stimulants including phorbol esters, tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin-1 (43, 44, 45). Stimulation triggers the release of NF-{kappa}B from I{kappa}B, resulting in the translocation of its subunits, p50 and p65, into the nucleus. The subunits of NF-{kappa}B are now known to form homodimers or heterodimers, interact with subunits from the fos and jun protooncogene families, and interact with other members of the rel family to regulate the transcription of multiple genes involved in immune and growth responses of cells (42, 43, 44, 45). Thus, they are implicated in cytokine activation (interleukins-1, -6, and -8, and interferon-ß, for example), cell adhesion (regulation of endothelial-leukocyte adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1), and T cell growth, as well as commitment. In short, they are implicated in the expression of a pleiotropic array of genes induced in response to such agents as growth factors, mitogens, tumor promoters, cytokines, TNF-{alpha}, and antioxidants, i.e. genes that are linked to both immunomodulation as well as growth regulation. It is, therefore, not surprising, in retrospect, that high concentrations of iodide would have both immunomodulatory actions and autoregulatory effects on the growth and function of the thyroid cell.

In studies of lymphocytes, sodium salicylate has been reported to inhibit the activation of NF-{kappa}B by interfering with the pathway that leads to the phosphorylation or degradation (or both) of I{kappa}B, i.e. its action is suggested to be at the level of the release of NF-{kappa}B from I{kappa}B and is opposite to the action of a phorbol ester such as TPA (46). In those studies (46), the sodium salicylate action was not duplicated by indomethacin or ETYA; hence the involvement of the phosphoinositide-Ca++-arachidonate system was excluded. This is not the case for iodide in the present study in thyrocytes. Thus, iodide would appear to have a direct transcriptional effect resulting from its activation of the phosphoinositide-Ca++-arachidonate pathway in thyroid cells; and the action of salicylates may be more complex than perceived in the earlier pioneering experiments (46). These data may reflect the importance of this pathway to the growth and function of thyrocytes (22, 23, 26, 27); additionally, the complexity of changes in p50 and p65 complexes noted in this report, i.e. decreases in Mod-1, increases in the p50/p65 heterodimer, but no change in the p50 homodimer, would not likely be caused simply by a change in NF-{kappa}B release from I{kappa}B and a mass action effect. A similar case can be made for the effect of the phorbol esters, i.e. the possibility must be considered that the action of TPA is more diverse than simply mediating the release of NF-{kappa}B from I{kappa}B.

The action of iodide to suppress class I and ATD, autoregulate thyroid growth, and act via NF-{kappa}B, with its pleiotropic effects on genes implicated in both the immune and growth response of the cell, also raises the specter of tissue-specific control. Thus, iodide appears to represent a thyroid-specific action on a ubiquitous regulatory system, since its effect is lost in the FRT thyroid cell, whose thyroid function is lost coincident with the loss of a thyroid-restricted transcription factor, thyroid transcription factor-1 (7). This cannot be related solely to the loss of the TSH-induced iodide symporter, since iodide action on class I is still evident in 5H medium in which TSH is not present. Continued study of the action of iodide on the NF-{kappa}B system of the thyroid may, therefore, contribute to our understanding of the tissue-specific controls on this ubiquitous regulatory system.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Highly purified bovine TSH used in experiments was obtained from the hormone distribution program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH (NIDDK-bTSH; 30 U/mg). Hydrocortisone was from Calbiochem (La Jolla, CA); insulin, TPA, ASA, indomethacin, and ETYA were from the Sigma Chemical Co. (St. Louis, MO). [{alpha}-32P]deoxy-CTP (3000 Ci/mmol), [14C]chloramphenicol (50 mCi/mmol), and [{gamma}-32P]ATP were from Amersham (Arlington Heights, IL). The p50 subunit of NF-{kappa}B was from Promega (Madison, WI); antibodies to the p50 and p65 subunits of NF-{kappa}B, c-rel, the c-fos family, and c-jun were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The NF-{kappa}B consensus binding site, 5'-AGTTGAGGGGACTTTCCCAGGC-3', was from Promega; the 24 mer, 5'-CGGTGGTGGGGAGTCCCCGTGTCC-3', encompassing enhancer A, as well as its mutants, was from Operon Technologies, Inc. (Alameda, CA).

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 (7, 21, 24, 25 25A, 28, 47, 48, 49). Thus, their doubling time with TSH was 36 ± 6 h; and without TSH, they did not proliferate. After 6 days in medium with no TSH, addition of 1 x 10-10 M TSH resulted in 10-fold or better increases in thymidine incorporation into DNA. Fresh cells were diploid and between their fifth and 20th passage. FRTL-5 thyroid cells with an aged phenotype had been passaged 30 or more times (26). Like fresh phenotype cells, they were diploid and did not proliferate without TSH. However, in the absence of TSH, their basal thymidine incorporation into DNA during 72 h was increased at least 10-fold over fresh phenotype cells and, after 6 days in medium with no TSH, addition of 1 x 10-10 M TSH resulted in only an approximately 2-fold increase in thymidine incorporation into DNA. Their doubling time with TSH was about 26 ± 6 h.

All cells were grown in 6H medium consisting of Coon’s modified F12 (Sigma Chemical Co.) supplemented with 5% calf serum, 1 mM nonessential amino acids (GIBCO, Grand Island, NY), 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) (48, 49). 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
Total cellular RNA was isolated, Northern analyses were performed, and filters were sequentially hybridized with the following cDNA probes (0.5–1.0 x 106 cpm/ml) (7, 25 25A, 28, 50): a 1.0-kb HpaI fragment of the MHC class I clone spanning the entire cDNA insert (7, 25 25A, 28) and ß-actin, which was provided by Dr. B. Paterson, National Cancer Institute, NIH.

Construction of MHC Class I Promoter-CAT Chimeric Plasmids
The CAT chimeras of the PD1 swine 5'-flanking sequences, p(-1100)CAT, p(-549)CAT, p(-203)CAT, and p(-127)CAT have been described (28, 47); they are numbered from the nucleotide at the 5'-end to +1 bp, the start of transcription (28, 47). CAT constructs with mutated enhancer A sequences were created by two-step, recombinant PCR methods (28, 51, 52). In the first step, two PCR products that overlap the sequence were created, both of which contain the same mutation introduced as part of the PCR primers. The second step PCR was performed using these overlapped PCR products as template and DNA sequence of the 5'- or 3'-end of the final products as primer. The PCR products were inserted into the multicloning site of pSV3CAT (28). A Perkin-Elmer Cetus (Norwalk, CT) thermal cycler was used; the reaction was performed at 94 C (1 min), 55 C (2 min), and 72 C (3 min) for 30 cycles; final extension was for 7 min at 72 C. The amplified fragment was purified using 1.5% agarose gel electrophoresis.

The sequences of all constructs were confirmed by a standard method (53); DNA was prepared and twice purified by CsCl gradient centrifugation (54).

Transfection
FRTL-5 cells stably transfected with class I promoter-CAT chimeras have been described (28). To test the effect of iodide, cells were grown to 70–80% confluency in 6H medium and then maintained without TSH (5H medium) for 6 days, at which time they were exposed to 1 mM iodide for 24 h before CAT activity was measured. Transient transfections used the same class I-CAT chimeras and a diethylaminoethyl (DEAE)-dextran procedure (28, 55). Cells were grown to near (80%) confluency in 6H medium, shifted to 5H medium for 1 day, washed twice with Dulbecco’s modified PBS (DPBS), pH 7.4, and incubated 1 h with 5 ml serum-free 5H medium containing the plasmid DNA plus 250 µg DEAE-dextran (5 Prime->3 Prime, Inc., Boulder, CO). Cells were then exposed to 10% dimethylsulfoxide in DPBS for 3 min, washed twice in DPBS, cultured in 5H medium for 48 h, and then maintained therein another 24 h with or without iodide as noted. Efficiency of transfection was determined by cotransfection with 5 µg pRSVLuc, kindly provided by Dr. S. Subramani, University of California (La Jolla, CA) (56). CAT values, mean ± SE of three experiments, are normalized to luciferase activity and protein using the Promega assay system and a Moonlight 2010 luminometer (28). Cell viability was approximately 80% in all experiments. CAT assays were performed as described (28, 47, 57).

Cell and Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium (+TSH) until 80% confluent and then maintained in 5H medium (-TSH) with 5% calf serum. Cells were exposed to iodide or TPA, in the presence or absence of ASA, indomethacin, or ETYA at the concentrations noted. Unless otherwise noted, cellular extracts were prepared by a modification of a described method (28, 47, 58) after scraping and centrifuging (500 x g) cells that had been washed twice in cold DPBS, pH 7.4. The pellet was resuspended in 2 vol of Dignam buffer C (80) [25% glycerol, 20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.5 mM dithiothreitol (DTT), 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.5 mM phenyl-methylsulfonyl fluoride (PMSF)]. The final NaCl concentration was adjusted on the basis of cell pellet volume to 0.42 M. Cells were lysed by seven repeated cycles of freezing and thawing. The extracts were centrifuged at 32,500 rpm (100,000 x g) and at 4 C for 20 min. The supernatant was recovered, aliquoted, and stored at -70 C.

Nuclear extracts were prepared as described (28, 59, 60) from identically treated and harvested FRTL-5 cells. After centrifugation at 500 x g, the cells were suspended in five 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 DTT, 0.5 mM PMSF, 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 PMSF, 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.

EMSAs
Oligonucleotides used for EMSA were synthesized or were purified from 2% agarose gel using QIAEX (Quiagen, Chatsworth, CA) after restriction enzyme treatment of the chimeric CAT constructs. They were labeled with [{alpha}-32P]dCTP using Klenow or with [{gamma}-32P]ATP using T4 polynucleotide kinase and purified on an 8% native polyacrylamide gel (28, 47, 61). EMSAs were performed as previously described (28, 47) but used two different binding conditions. Binding reactions in low salts and the absence of detergents were carried out in a volume of 20 µl for 30 min at room temperature. The reaction mixtures contained 1.5 fmol [32P]DNA, 2 µg cell or nuclear extract, and 0.5 µg poly(dI-dC) in 10 mM Tris-Cl at pH 7.9, 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol. Alternatively, binding reactions were performed in high salts plus detergent and included 1.5 fmol of [32P]DNA, 2 µg nuclear extract, and 0.5 µg poly(dI-dC) in 10 mM Tris-Cl at pH 7.9, 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol. Where indicated, unlabeled oligonucleotides were added to the binding reaction as competitors and incubated with the extract for 20 min before the addition of labeled DNA. In experiments using antiserum, extracts were incubated in the same buffer containing antiserum or normal rabbit serum at 20 C for 1 h before being processed. After incubations, reaction mixes were subjected to electrophoresis on 5% native polyacrylamide gels at 160 V in 0.5xTris-borate-EDTA and at room temperature. Gels were dried and autoradiographed.

Other Assays
Protein concentration was determined by Bradford’s method (Bio-Rad) and used recrystallized BSA as the standard.

Statistical Significance
All experiments were repeated at least three times with different batches of cells. Values are the mean ± SE of these experiments where noted. Significance between experimental values was determined by two-way ANOVA and are significant if P values were <0.05 when data from all experiments were considered.


    FOOTNOTES
 
Address requests for reprints to: Dr. Leonard D. Kohn, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Building 10, Room 9C101B, National Institutes of Health, Bethesda, Maryland 20892-1360. E-mail: lenk{at}bdg10.niddk.nih.gov

V. M., C. G., and M. S. were supported by the Interthyr Research Foundation, 301 St. Paul Place, Suite 712, Baltimore, MD 21202, during this project. M. S. was also supported by a Thyroid Research Council Award from the Knoll Pharmaceutical Company, 3000 Continental Drive-North, Mount Olive, NJ 07828, during a portion of this project.

1 Current Address: 1st Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan. Back

2 Current address: Department of Internal Medicine, Chungnam National University Hospital, 640 Daesa-Dong, Chung-ku, Daejon, 301–040, Korea. Back

3 Current address: Cattedra di Endocrinologia, Università degli Studi "G. D’Annunzio" - Chieti, Faculty of Medicine and Surgery, Palazzina Scuole di Specializzazione, Via dei Vestini, 66100 Chieti, Italy. Back

4 Current address: Department of Surgery, Johns Hopkins University, Ross Building, Room 756, 720 Rutland Avenue, Baltimore, Maryland 21287. Back

Received for publication April 18, 1997. Revision received October 21, 1997. Accepted for publication October 23, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Singer DS, Maguire JE 1990 Regulation of the expression of Class I MHC genes. CRC Crit Rev Immunol 10:235–257
  2. Ting JP-Y, Baldwin AS 1993 Regulation of MHC gene expression. Curr Opin Immunol 5:8–16[Medline]
  3. Kohn LD, Giuliani C, Montani V, Napolitano G, Ohmori M, Ohta M, Saji M, Schuppert F, Shong M-H, Suzuki K, Taniguchi S-I, Yano K, Singer DS 1995 Antireceptor immunity. In: Rayner D, Champion B (eds) Thyroid Immunity. Landes, Austin, TX, pp 115–170
  4. Todd I, Londei M, Pujol-Borrell R Mirakian R, Feldmann M, Bottazzo GF 1986 HLA/DR expression on epithelial cells: the finger on the trigger. Ann NY Acad Sci 475:241–249[Abstract]
  5. Bottazzo GF, Dean BM, McNally JM, et al. 1985 In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 313:353–360[Abstract]
  6. Hokland M, Heron I, Hokland P, Basse P, Berg K 1986 Measurements of changes in histocompatibility antigens induced by interferons. Methods Enzymol 119:688–693[Medline]
  7. Saji M, Moriarty J, Ban T, Singer DS, Kohn LD 1992 MHC Class I expression in rat thyroid cells is regulated by hormones, methimazole, and iodide, as well as interferon. J Clin Endocrinol Metab 75:871–878[Abstract]
  8. Burman P, Totterman TH, Oberg K, Karlson FA 1986 Thyroid autoimmunity in patients on long term therapy with leukocyte-derived interferon. J Clin Endocrinol Metab 63:1086–1090[Abstract]
  9. Feintiman IS, Balkwill FR, Thomas BS, Russel MJ, Todd I, Bottazzo GF 1988 An autoimmune aetiology for hypothyroidism following interferon therapy for breast cancer. Eur J Cancer Clin Oncol 24:1299–1303[Medline]
  10. Mayet W-J, Hess G, Gerken G, Rossol S, Voth R, Manns M, Meyer zum Buschenfelde K-H 1989 Treatment of chronic type B hepatitis with recombinant {alpha}-interferon induces autoantibodies not specific for autoimmune chronic hepatitis. Hepatology 10:24–28[Medline]
  11. Mozes E, Kohn LD, Hakim F, Singer DS 1993 Mice deficient in expression of MHC class I are resistant to experimental systemic lupus erythematosus. Science 261:91–93[Medline]
  12. Taki T, Nagata M, Ogawa W, Hatamori N, Hayakawa M, Hari J, Shii K, Baba S, Yokono K 1991 Prevention of cyclophosphamide-induced and spontaneous diabetes in NOD/Shi/Kbe mice by anti MHC class I Kd monoclonal antibody. Diabetes 40:1203–1209[Abstract]
  13. Serreze DV, Leiter EH, Christianson GJ, Greiner D, Roopenian DC 1994 Major histocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis resistant. Diabetes 43:505–509[Abstract]
  14. Chan C-C, Gery I, Kohn LD, Nussenblatt RB, Mozes E, Singer DS 1995 Periocular inflammation in mice with experimental systemic lupus erythematosus (SLE): a new experimental blepharitis and its modulation. J Immunol 154:4830–4835[Abstract/Free Full Text]
  15. Singer DS, Kohn LD, Zinger H, Mozes E 1994 Methimazole can prevent development of disease in an experimental model of systemic lupus erythematosus. J Immunol 153:873–880[Abstract/Free Full Text]
  16. Cooper DS 1996 Treatment of thyrotoxicosis. In: Braverman LE, Utiger RD (eds) Werner and Ingbar’s the Thyroid: A Fundamental and Clinical Text. Lippincott-Raven, Philadelphia, pp 713–734
  17. Nagataki S, Yokoyama N 1996 Autoregulation: effects of iodide. In: Braverman LE, Utiger RD (eds) Werner and Ingbar’s the Thyroid: A Fundamental and Clinical Text. Lippincott-Raven, Philadelphia, pp 241–247
  18. Becks GP, Eggo MC, Burrow GN 1988 Organic iodine inhibits deoxyribonucleic acid synthesis and growth in FRTL-5 thyroid cells. Endocrinology 123:545–551[Abstract]
  19. Saji M, Isozaki O, Tsushima T, Arai M, Miyakawa M, Ohba Y, Tsuchiya Y, Sano T, Shizume K 1988 Inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells. Acta Endocrinol (Copenh) 119:145–151[Medline]
  20. Tramontano D, Veneziani BM, Lombardi A, Villone G, Ingbar SH 1989 Iodine inhibits the proliferation of rat thyroid cells in culture. Endocrinology 136:269–282[Abstract]
  21. Schuppert F, Taniguchi S-I, Schröder S, Dralle H, von zur Mühlen A, Kohn LD 1996 In vivo and in vitro evidence for iodide regulation of MHC class I and class II expression in human thyroid disease. J Clin Endocrinol Metab 81:3622–3628[Abstract]
  22. Ekholm R, Kohn LD, Wollman S 1989 Control of the thyroid gland: regulation of its normal function and growth. Adv Exp Med Biol 261:1–403
  23. Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor. Vitam Horm 50:287–384[Medline]
  24. Saji M, Kohn LD 1990 Effect of hydrocortisone on the ability of thyrotropin to increase deoxyribonucleic acid synthesis and iodide uptake in FRTL-5 rat thyroid cells: opposite regulation of adenosine 3',5'-monophosphate signal action. Endocrinology 127:1867–1876[Abstract]
  25. Saji M, Moriarty J, Ban T, Kohn LD, Singer DS 1992 Hormonal regulation of MHC class I genes in rat thyroid FRTL-5 cells: TSH induces a cAMP-mediated decrease in Class I expression. Proc Natl Acad Sci USA 89:1944–1948[Abstract]
  26. Taniguchi S-I, Giuliani C, Saji M, Napolitano G, Singer DS, Kohn LD, Transcriptional regulation of major histocompatibility (MHC) class I gene expression in thyroid cells by iodide involves enhancer A and the transcription factor, NF-{kappa}B. Program of the 77th annual meeting of The Endocrine Society, Washington DC, 1995 (Abstract OR24–3), p 77
  27. Bellur S, Tahara K, Saji M, Grollmann EF, Kohn LD 1990 Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA. Endocrinology 127:1526–1540[Abstract]
  28. Tahara K, Grollman EF, Saji M, Kohn LD 1991 Regulation of prostaglandin synthesis by thyrotropin, insulin, or insulin-like growth factor-I, and serum in rat FRTL-5 thyroid cells. J Biol Chem 266:440–448[Abstract/Free Full Text]
  29. Giuliani C, Saji M, Napolitano G, Palmer LA, Taniguchi S-I, Shong M, Singer DS, Kohn LD 1995 Hormonal modulation of MHC class I gene expression involves an Enhancer A-binding complex consisting of fra-2 and the p50 subunit of NF-{kappa}B. J Biol Chem 270:11453–11462[Abstract/Free Full Text]
  30. Taniguchi S-I, Montani V, Giuliani C, Saji M 1995 Iodide and TPA can decrease major histocompatibility (MHC) class I gene expression in thyroid cells via NF-{kappa}B, but they use different phospholipase c (PLC)-activated signal pathways. Thyroid [Suppl 1] 5:S-67
  31. Corvilain B, Laurent E, Lecomte M, VanSande J, Dumont JE 1994 The role of cyclic adenosine 3', 5'-monophosphate and the phosphoinositol-Ca++ cascades in mediating the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices. J Clin Endocrinol Metab 79:152–159[Abstract]
  32. Schuppert F, Reiser M, Heldin NE, et al 1994 TSH receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by Northern blot analysis and in situ hybridization. Acta Endocrinol (Copenh) 131:480–488
  33. 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]
  34. Todd I, Pujol-Borrell R, Hammond LJ, Bottazzo GF, Feldmann M 1985 Interferon-gamma induces HLA-DR expression by thyroid epithelium. Clin Exp Immunol 61:265–273[Medline]
  35. 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]
  36. Misaki T, Tramontano D, Ingbar S 1988 Effects 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]
  37. Zakarija M, Hornicek FJ, Levis S, Mckenzie JM 1988 Effects of gamma interferon and tumor necrosis factor alpha on thyroid cells: induction of class II antigen and inhibition of growth stimulation. Mol Cell Endocrinol 58:329–336
  38. Wolff J, Chaikoff IL 1948 Plasma inorganic iodide as a homeostatic regulator of thyroid function. J Biol Chem 174:555–568[Free Full Text]
  39. Sarne DH, DeGroot LJ 1989 Hypothalamic and Neuroendocrine regulation of thyroid hormone. In: DeGroot LJ (ed) Endocrinology. WB Saunders Co, Philadelphia, vol 1:574–589
  40. Grollman EF, Smolar A, Ommaya A, Tombaccini D, Santisteban P 1986 Iodine suppression of iodide uptake in FRTL-5 thyroid cells. Endocrinology 118:2477–2482[Abstract]
  41. Nishina H, Sato H, Suzuki T, Sato M, Iba H 1990 Isolation and characterization of fra-2, an additional member of the fos gene family. Proc Natl Acad Sci USA 87:3619–3623[Abstract]
  42. Stein B, Cogswell PC, Baldwin Jr AS 1993 Functional and physical associations between NF-{kappa}B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol 13:3964–3974[Abstract]
  43. Korber B, Hood L, Stroynowski I 1987 Regulation of murine class I genes by interferons is controlled by regions located both 5' and 3' to the transcription initiation site. Proc Natl Acad Sci USA 84:3380–3384[Abstract]
  44. Van’t Veer LJ, Beijersbergen RL, Bernards R 1993 N-myc suppresses major histocompatibility complex class I gene expression through down regulation of the p50 subunit of NF-{kappa}B. EMBO J 12:195–200[Abstract]
  45. Grilli M, Chiu J-S, Lenardo MJ 1993 NF-{kappa}B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol 143:1[Medline]
  46. Stein B, Baldwin Jr AS, Ballard DW, Greene WC, Angel P, Herrlich P 1993 Cross-coupling of the NF-{kappa}B p65 and Fos/Jun transcription factors produces potentiated biological responses. EMBO J 12:3879–3891[Abstract]
  47. Nolan GP 1994 NF-AT-AP-1 and Rel-bZip: hybrid vigor and binding under the influence. Cell 77:795–798[Medline]
  48. Kopp E, Gosh S 1994 Inhibition of NF-{kappa}B by sodium salicylate and aspirin. Science 265:956–959[Medline]
  49. Saji M, Shong M, Napolitano G, Palmer LA, Taniguchi S-I, Ohmori M, Ohta M, Suzuki K, Kirshner SL, Giuliani C, Singer DS, Kohn LD 1996 Regulation of major histocompatibility complex class I gene expression in thyroid cells: role of the cAMP response element-like sequence. J Biol Chem 272:20096–20107[Abstract/Free Full Text]
  50. Ambesi-Impiombato FS 1986 Fast-growing thyroid cell strain. U.S. Patent 4,608,341
  51. Kohn LD, Valente WA, Grollman EF, Aloj SM, Vitti P 1986 Clinical determination and/or quantification of thyrotropin and a variety of thyroid stimulatory or inhibitory factors performed in vitro with an improved thyroid cell line FRTL-5. US Patent no. 4,609,622
  52. Isozaki O, Kohn LD, Kozak CA, Kimura S 1989 Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells. Mol Endocrinol 3:1681–1692[Abstract]
  53. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA 1988 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491[Medline]
  54. Higuchi R 1990 Recombinant PCR In: Innis MA, Gelfand DH, Sninsky JI, White TJ (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, pp 177–183
  55. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467[Abstract]
  56. Davis LG, Dibner MD, Battey JF 1986 Basic Methods in Molecular Biology, Elsevier, New York, pp 93–98
  57. Lopata MA, Cleveland DW, Sollner-Webb B 1984 High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 12:5707–5717[Abstract]
  58. De Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Medline]
  59. Gorman CM, Moffat LF, Howard BH 1982 Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–1051[Medline]
  60. Dignam J, Lebovitz R, Roeder R 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract]
  61. Ikuyama S, Niller HH, Shimura H, Akamizu T, Kohn LD 1992 Characterization of the 5'-flanking region of the rat thyrotropin receptor gene. Mol Endocrinol 6:793–804[Abstract]
  62. Ikuyama S, Shimura H, Hoeffler JP, Kohn LD 1992 Role of the cyclic adenosine 3',5'-monophosphate response element in efficient expression of the rat thyrotropin receptor promoter. Mol Endocrinol 6:1701–1715[Abstract]
  63. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY