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Endocrinology Vol. 139, No. 1 280-289
Copyright © 1998 by The Endocrine Society

ARTICLES

Major Histocompatibility Class II HLA-DR{alpha} Gene Expression in Thyrocytes: Counter Regulation by the Class II Transactivator and the Thyroid Y Box Protein

Valeria Montani1, Shin-ichi Taniguchi2, Minho Shong3, Koichi Suzuki, Masayuki Ohmori4, Cesidio Giuliani1,5, Giorgio Napolitano5, Motoyasu Saji1, Bruno Fiorentino, Andreas M. Reimold1, Jenny P.-Y. Ting, Leonard D. Kohn and Dinah S. Singer

Cell Regulation Section (V.M., S.-i.T., M.S., K.S., M.O., C.G., G.N., B.F., 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; Department of Surgery (M.S.), Johns Hopkins University, Baltimore, Maryland 21287; Departments of Cancer Biology and Medicine (A.M.R.), Harvard School of Public Health and Harvard Medical School, Boston, Massachusetts 02115; and Lineberger Comprehensive Cancer Research Center (J.P.-Y.T.), University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aberrant expression of major histocompatibility complex (MHC) class II proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by {gamma}-interferon ({gamma}-IFN). To define elements and factors that regulate class II gene expression in thyrocytes and that might be involved in aberrant expression, we have studied {gamma}-IFN-induced HLA-DR{alpha} gene expression in rat FRTL-5 thyroid cells. The present report shows that class II expression in FRTL-5 thyrocytes is positively regulated by the class II transactivator (CIITA), and that CIITA mimics the action of {gamma}-IFN. Thus, as is the case for {gamma}-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DR{alpha} gene, the S, X1, X2, and Y boxes between -137 to -65 bp, are required for class II gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and {gamma}-IFN do not cause additive increases in HLA-DR{alpha} gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the {gamma}-IFN pathway to increased class II gene expression. Additionally, {gamma}-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of {gamma}-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both {gamma}-IFN and CIITA to increase class II gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC class I gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC class II expression in human glioma cells. We conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the {gamma}-IFN-regulated transduction system which, respectively, increase or decrease class II gene expression in thyrocytes and may, therefore, be involved in aberrant class II expression associated with autoimmune thyroid disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ABNORMAL or aberrant major histocompatibility complex (MHC) class II gene expression is associated with autoimmune thyroid disease (ATD) (1, 2, 3). A recent report supports the importance of aberrant class II expression in the development of ATD. Thus, immunizing mice with fibroblasts transfected with the human TSH receptor (TSHR) and a MHC class II molecule, but not either alone, has induced ATD with the major humoral and histological features of Graves’ disease (4). These results support the view that acquisition of antigen-presenting ability on a thyroid cell, as a result of aberrant class II expression, can activate T and B cells normally present in an animal and allow normal immune tolerance to be broken. Understanding the mechanisms underlying the basis for aberrant class II expression in thyrocytes seems, therefore, to be important to understanding how ATD might develop.

{gamma}-Interferon ({gamma}-IFN) can induce class II antigen expression in FRTL-5 thyroid cells and mimic changes in human thyrocytes seen in ATD (7, 8, 9, 10). We therefore initiated a study of {gamma}-IFN-induced HLA-DR{alpha} gene expression in rat FRTL-5 thyroid cells, as a model to define elements and factors that might be important in ATD. In a separate study (11, 12), we showed that the ability of {gamma}-IFN to induce aberrant HLA-DR{alpha} gene expression in FRTL-5 thyroid cells required, like antigen-presenting cells of the immune system (5, 6, 13, 14, 15), the highly conserved S, X1, X2, and Y boxes on the DR{alpha} 5'-flanking region, -137 to -65 bp. We additionally showed (12) that {gamma}-IFN-induced aberrant expression was associated with increased formation of a specific and novel protein/DNA complex containing CBP, a coactivator of cAMP response element-binding proteins (16); and that {gamma}-IFN-induced formation of this complex, as well as increased HLA-DR{alpha} gene expression, was suppressed by methimazole (11, 12), an agent effective in treating Graves’ disease and preventing experimental thyroiditis in rats or mice (17, 18, 19).

Two factors known to regulate class II gene expression in immune cells are the class II transactivator (CIITA) and a Y box-binding protein (5, 6). CIITA is a non-DNA-binding protein transactivator that functions as a molecular switch to control constitutive and inducible MHC class II gene expression in immune cells; CIITA expression is induced by {gamma}-IFN and is believed to be involved in its activity (6, 20, 21, 22, 23, 24, 25). The human Y box protein, YB-1, was cloned based on its ability to bind to the Y box, an inverted CCAAT box, of the MHC class II gene (26) and has been shown to suppress HLA-DR{alpha} gene expression in human glioblastoma cells (27, 28). The present report evaluates the role of CIITA and the Y box-binding protein in {gamma}-IFN-induced HLA-DR{alpha} gene expression in FRTL-5 thyrocytes and in the ability of {gamma}-IFN to induce the formation of this novel, methimazole-sensitive complex. Rather than YB-1, we use a Y box protein cloned from FRTL-5 thyroid cells based on its ability to suppress TSHR gene expression (29) and therefore termed TSHR suppressor element binding protein-1 (TSEP-1). TSEP-1 is involved in TSH/cAMP and methimazole suppression of MHC class I gene expression in FRTL-5 cells (30, 31, 32).

The present report shows that CIITA and the thyrocyte Y box protein, TSEP-1, are important factors controlling {gamma}-IFN-induced aberrant class II gene expression in thyrocytes and are important in the formation of the {gamma}-IFN-induced novel complex in these cells. We suggest, therefore, they are involved in aberrant class II expression associated with ATD. The data support the conclusion that the negative regulation of class II, as well as the class I and TSHR genes, involves common transcription factors. They are consistent with our hypothesis (31, 32) that coordinate negative control of class II, class I, and the TSHR genes by common transcription factors is necessary to maintain self-tolerance during hormone-induced increases in thyroid cell growth and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Highly purified bovine TSH was obtained from the hormone distribution program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIDDK-bTSH I-1; 30 U/mg) or was a previously described preparation, 26 ± 3 U/mg, with the molecular weight and amino acid and carbohydrate composition of purified TSH (33). Rat recombinant {gamma}-IFN was from GIBCO BRL (Life Technologies, Inc), Gaithersburg, MD; [14C]Chloramphenicol (50 mCi/mmol) was from DuPont-New England Nuclear (Boston, MA); [{gamma}-32P]ATP (6000 Ci/mmol) was from Amersham (Arlington Height, IL). The following antisera from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) were used: CREB-1 (C-21), sc-186; CREB-2 (C-20), sc-200; activating transcription factor (ATF)-2 (C-19), sc187; ATF-3 (C-19), sc-188; ATF-1 (FI-1) sc-241; ATF-4 (Z-5), sc-244; CBP (A22), sc-369; p65 (A) G, sc-109G; and p50 (NLS) G, sc-114G. The source of all other materials was Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.

Plasmids
The HLA-DR{alpha} promoter constructs used herein were obtained from Dr. L. H. Glimcher (Harvard School of Public Health and Department of Medicine, Harvard University Medical School, Boston, MA); their construction and characteristics have been described previously (15). The Y box expression vector, pRcCMV-TSEP-1, was constructed as described in an earlier report (29). The source of the human CIITA cDNA has been described (34); it is clone CIITA-8, which is identical to CIITA as originally described (20), but has an additional 20 bp of 5'- and 1.6 kb of 3'-untranslated sequence (34). For use in the present studies we ligated the full-length clone into pcDNA3 (Invitrogen, San Diego, CA) and termed it pCIITA.

Cell culture
FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD; ATCC CRL8305) were a fresh subclone (F1) with the properties described previously (35, 36). They were grown in Coon’s modified F-12 medium containing 5% heat-treated, mycoplasma-free calf serum (GIBCO), 1 mM nonessential amino acids (GIBCO), and a mixture of six hormones (6H) containing 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). Cells were diploid and between their fifth and 25th passage. Fresh medium was added every 2 or 3 days, and cells were passaged every 7–10 days. In some experiments, as noted, cells were grown to near confluency in 6H medium and then maintained in 5H medium, which contains no TSH, for 6 days before experiments were initiated.

Transient expression analysis
Transient transfections in FRTL-5 cells were performed as described (12, 31, 37), using one of the following procedures. In the first, cells were cultivated in 6H medium to approximately 80% confluency, harvested, washed, and resuspended (1.5 x 107 cells/ml) in 0.85 ml electroporation buffer (272 mM sucrose, 7 mM sodium phosphate buffer, pH 7.4, and 1 mM MgCl2). Plasmid DNA was added, 20 µg of the chloramphenicol acetyltransferase (CAT) chimera together with 2 µg pRSV-luciferase, which is used to measure the efficiency of transfection (38). Cells were pulsed (330 V; capacitance 25 µfarad), plated (6 x 106 cells per 10-cm dish), and cultured in 6H medium plus 5% calf serum supplemented or not with {gamma}-IFN. At the times noted, cells were harvested for CAT and luciferase assays. The second procedure differed as follows. FRTL-5 cells were grown to 80% confluency in 6H medium and then maintained 6 days in 5H medium plus 5% calf serum. Cells were returned to 6H medium for 12 h and transfected as described above. Twelve hours later the medium was changed to fresh 5H medium with 5% calf serum supplemented or not with {gamma}-IFN. Cell viability was approximately 80% in all experiments.

Cotransfections with pCIITA, pRcCMV-TSEP-1, or their respective vector-only controls, pcDNA3 or pRcCMV, used 20 µg DNA unless otherwise noted. CAT activity was measured as described previously (31, 37, 39). CAT values, mean ± SE of three experiments, are normalized to luciferase activity using the Promega (Madison, WI) assay system.

Cellular extracts
Cell extracts were made by a modification of the method of Dignam et al. (12, 31, 37, 40). Briefly, FRTL-5 cells were harvested by scraping after being washed twice in ice-cold PBS and pelleted. The pellet was resuspended in 2 vol of Dignam buffer C (20 mM HEPES at pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 25% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin). 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 at 100,000 x g and at 4 C for 20 min. The supernatant was recovered, aliquoted, and stored at -70 C.

Electrophoretic mobility shift assays (EMSA)
Oligonucleotides used for EMSA were synthesized (Operon Technologies, Inc., Alameda, CA) or were purified from 2% agarose gel using QIAEX (Qiagen, Chatsworth, CA) or Jet-Sorb (Genemed, Frederick, MD), following restriction enzyme treatment of the chimeric class II CAT constructs. They were labeled with [{alpha}-32P]deoxycytosine triphosphate using Klenow or with [{gamma}-32P]ATP using T4 polynucleotide kinase and then purified on an 8% native polyacrylamide gel (12, 31, 37, 41, 42, 43).

EMSAs were performed basically as previously described (12, 31, 37, 42, 43). Binding reactions were carried out in a volume of 20 µl for 30 min at room temperature. The reaction mixtures contained 1.5 fmol of [32P]DNA, 3 µg cell extract, and 1.5 µg poly(deoxyinosinic-deoxycytidylic)acid in 10 mM Tris-Cl at pH 7.9, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol. After incubations, reaction mixtures were subjected to electrophoresis on 3.5% or 5% native polyacrylamide gels at 160 V in 0.5xTris-borate-EDTA, at room temperature, for 1.5–2.0 h. Gels were dried and autoradiographed at -80 C overnight unless otherwise noted.

Other methods
Protein concentration was determined by Bradford’s method (Bio-Rad Laboratories, Richmond, CA); recrystallized BSA was the standard. DNA was prepared and purified by CsCl gradient centrifugation (44). The sequences of all constructs were confirmed by a standard method (45). RNA was isolated and Northern analyses performed as described (46). The CIITA probe was residues 5–1395 of the nucleotide sequence (34); rat ß-actin was provided by Dr. B Paterson (National Cancer Institute, Bethesda, MD). Radiolabeling of all probes, hybridization (1.0 x 106 cpm/ml), and washing were described previously (46).

Statistical significance
All experiment 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of HLA-DR{alpha} in FRTL-5 thyroid cells is increased by CIITA as well as {gamma}-IFN; the same 5'-promoter elements are required by each
The HLA-DR{alpha} -176-bp minimal promoter, coupled to the CAT reporter gene, is not expressed in transiently transfected FRTL-5 thyrocytes unless the cells are treated with rat recombinant {gamma}-IFN (Ref. 12; Fig. 1Go). In the absence of {gamma}-IFN, cotransfection of pCIITA containing full-length human CIITA cDNA, but not pcDNA3, the vector incorporating the CIITA insert, can also significantly increase HLA-DR{alpha} CAT activity (Fig. 1Go). As was the case for rat recombinant {gamma}-IFN (12), CIITA transfection was associated with an increase in endogenous class II expression measured by flow cytometry (data not shown), i.e. its action on HLA-DR{alpha} appeared to reflect a coordinate effect on the endogenous class II antigen.



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  		Figure 1. Transfection of CIITA in FRTL-5 thyrocytes increases HLA-DR{alpha} expression, measured using the -176 bp DR{alpha}-CAT chimera. The -176-bp DR{alpha}-CAT chimera containing -176 to +45 bp of the 5'-flanking region of the HLA DR{alpha} gene is diagrammatically represented at the bottom of the figure. Locations and 5'-termini of the S, X1, X2, and Y boxes are noted. FRTL-5 cells were grown to near confluency in medium with TSH (6H medium) then transiently transfected with the -176-bp DR{alpha}-CAT chimera plus pRSV-luciferase as described in Materials and Methods. One group of transfected cells was treated with 100 U/ml {gamma}-IFN ({gamma}-IFN +), another not treated ({gamma}-IFN -). A separate batch of cells was cotransfected with the -176 bp DR{alpha}-CAT chimera, pRSV-luciferase, and a plasmid containing the full-length human CIITA cDNA, pCIITA, or with a control vector without the CIITA insert, pcDNA3; neither of these two groups was exposed to {gamma}-IFN. After 48 h, CAT activity was measured as described. Results are expressed relative to the activity of -176 DR{alpha}-CAT in the absence of {gamma}-IFN (first open bar), after CAT activities were corrected both for luciferase activity and cell protein. These corrections in all cases resulted in less than 5% change in activity. Results are the mean ±SD of three separate experiments performed on three different batches of cells. A single asterisk (*) denotes a statistically significant increase (P < 0.01) in DR{alpha} promoter activity induced by {gamma}-IFN or pCIITA.

 
Evaluation of progressive 5'-deletions of the -176-bp DR{alpha}-CAT chimera to -137, -122, -111, -97, and -38 bp showed that the CIITA-increased HLA-DR{alpha} promoter activity in FRTL-5 thyroid cells is abrogated once the S box, -137 to -122 bp, is removed (Fig. 2Go). This parallels data obtained with {gamma}-IFN in FRTL-5 cells (12). Additionally, and also similar to {gamma}-IFN in the FRTL-5 cells, CIITA induction of HLA-DR{alpha} CAT activity required not only the S, but also the X1, X2, and Y boxes. Thus, mutation of each element alone resulted in a loss in the increase effected by CIITA overexpression and a return toward levels present when the vector alone, pcDNA3, was the cotransfectant (Fig. 3Go). CIITA regulation of class II gene expression in FRTL-5 thyroid cells thus involves the same DR{alpha} 5'-flanking region elements as does {gamma}-IFN (12).



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  		Figure 2. Effect of CIITA transfection on class II expression in FRTL-5 thyroid cells measured using the minimal -176 bp DR{alpha}-CAT construct and 5'-deletions thereof. The 5'-deletions of -176 DR{alpha}-CAT are diagrammed on the bottom of the figure relative to the S, X1, X2, and Y boxes. FRTL-5 cells grown to near confluency in medium with TSH (6H medium) were cotransfected with the noted DR{alpha}-CAT chimera, pRSV-luciferase, and either pCIITA or pcDNA3 as described (Materials and Methods) and as in Fig. 1Go. CAT activity was measured 48 h later. Results are expressed relative to the DR{alpha} vector control transfected with pcDNA3 (first open bar), after CAT activities were corrected both for luciferase activity and cell protein. These corrections in all cases resulted in less than 5% change in activity. Results are the mean ±SD of three separate experiments performed on three different batches of cells. A single asterisk (*) denotes a statistically significant increase (P < 0.01) in DR{alpha} promoter activity induced by pCIITA.

 


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  		Figure 3. Effect of CIITA transfection on class II expression in FRTL-5 thyroid cells measured using the -176-bp DR{alpha}-CAT construct having mutations within the S, X1, X2, and Y boxes. At the top of the figure is a diagrammatic presentation of the -176-bp DR{alpha}-CAT chimera with the locations of the S, X1, X2, and Y boxes noted by black boxes; also presented are the mutations made in each box. FRTL-5 cells grown to near confluency in medium with TSH (6H medium) were cotransfected with the noted DR{alpha}-CAT chimera, pRSV-luciferase, and either pCIITA or its vector control pcDNA3 (Materials and Methods). CAT activity was measured 48 h later. Results are expressed relative to the DR{alpha} vector control cotransfected with the pcDNA3 vector control (first open bar), after CAT activities were corrected both for luciferase activity and cell protein. These corrections in all cases resulted in less than 5% changes in activity. Results are the mean ± SD of three separate experiments performed on three different batches of cells. A single asterisk (*) denotes a statistically significant increase (P < 0.01) in DR{alpha} promoter activity induced by pCIITA; two asterisks (**) denotes a statistically significant decrease (P < 0.02) in DR{alpha} promoter activity exhibited by a HLA-DR{alpha}-CAT construct with mutations in the S, X1, or X2 mutations; three asterisks (***) denote a statistically significant decrease (P < 0.01) in DR{alpha} promoter activity of the HLA-DR{alpha}-CAT construct with a Y box mutation.

 
{gamma}-IFN-induced HLA-DR{alpha} gene expression in FRTL-5 thyroid cells appears to be mediated by CIITA
{gamma}-IFN-increased class II gene expression is higher in TSH-treated FRTL-5 cells than in cells maintained without TSH after 48 h of {gamma}-IFN treatment (Fig. 4BGo vs. 4A; Refs. 7, 12). At 48 h, HLA-DR{alpha} gene expression is lower in cells transfected with pCIITA and maintained in the presence of TSH (Fig. 4BGo) than in cells transfected with pCIITA maintained in the absence of TSH (Fig. 4AGo). Despite this difference in the effect of TSH on the ability of CIITA or {gamma}-IFN to increase class II expression at 48 h, the effects of CIITA and {gamma}-IFN were not additive in cells maintained with (Fig. 4BGo) or without TSH (Fig. 4AGo). Further, the HLA-DR{alpha} CAT activity in either situation matched that induced by CIITA overexpression alone, even when it was the lower of the two activities (Fig. 4BGo). This is consistent with the possibility that CIITA is an intermediate factor in the {gamma}-IFN-mediated increase of class II gene expression (5, 6, 21, 23, 24), albeit not solely responsible for all the actions of {gamma}-IFN as evidenced by differences in the influence of TSH on each of their activities. Support for the conclusion that CIITA is an intermediate in the action of {gamma}-IFN on class II expression in FRTL-5 thyroid cells is provided by the following experiments.



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  		Figure 4. Effect of {gamma}-IFN plus CIITA transfection on HLA-DR{alpha} expression in FRTL-5 thyroid cells maintained in the absence (A) or presence (B) of TSH in the medium. In panel A, FRTL-5 cells were grown to 80% confluency in medium with TSH (6H), maintained 6 days in medium with no TSH (5H), then returned to 6H medium for 12 h before being transfected with -137-bp DR{alpha}-CAT together with pRSV-luciferase and pcDNA3 or pCIITA as noted. After 12 h, the medium was replaced with fresh medium without TSH and with or without 100 U/ml {gamma}-IFN. CAT activity was measured after 48 h. In Panel B, FRTL-5 cells were grown to 80% confluency in medium with TSH (6H), maintained 6 days in medium with no TSH (5H), then returned to 6H medium for 12 h before being transfected with -137 DR{alpha}-CAT together with pRSV-luciferase and either pcDNA3 or pCIITA as noted. Twelve hours after transfection, fresh medium was added with TSH and with or without 100 U/ml {gamma}-IFN; CAT activity was measured 48 h thereafter. After CAT activities were corrected both for luciferase activity and cell protein, results were expressed relative to the activity of the CAT-vector control in cells without TSH (first open bar in panel A). These corrections in all cases resulted in less than 5% changes in activity. Results are the mean ±SD of three separate experiments performed on three different batches of cells. In panels A and B a single asterisk (*) denotes a statistically significant increase (P < 0.01) in DR{alpha} CAT activity induced by {gamma}-IFN or pCIITA in cells maintained in the presence or absence of TSH. In panels A and B a double asterisk (**) denotes a statistically significant increase (P < 0.01) in DR{alpha} CAT activity induced by {gamma}-IFN and pCIITA in cells maintained in the presence or absence of TSH, but no significant additive effect of the two together and no significant difference from the effect of CIITA overexpression alone.

 
First, to ensure that CIITA could be an intermediate in the ability of {gamma}-IFN to activate class II gene expression in FRTL-5 thyrocytes, we examined the level of CIITA transcripts in FRTL-5 cells by Northern analysis. Under normal culture conditions, FRTL-5 cells do not express detectable levels of CIITA RNA (Fig. 5Go). However, treatment of FRTL-5 thyrocytes with 100 U/ml {gamma}-IFN for 48 h induced the appearance of messenger RNA (mRNA) transcripts that specifically interact with the human CIITA insert (Fig. 5Go). The multiplicity of CIITA transcripts has been noted in Raji cells, a Burkitt’s lymphoma B cell line (34).



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  		Figure 5. Effect of {gamma}-IFN on CIITA mRNA levels in FRTL-5 thyroid cells. FRTL-5 rat thyroid cells maintained in medium with 1 x 10-10 M TSH were treated with 100 U/ml {gamma}-IFN for 48 h. Total RNA was isolated and subjected (10 µg total/lane based on optical density) to Northern analysis using, sequentially, human CIITA and rat ß-actin probes.

 
Second, we tested whether CIITA overexpression could cause the same changes in protein/DNA complex formation with the DR{alpha} 5'-flanking region as did treatment with {gamma}-IFN: induction or increased formation of a faster migrating novel protein/DNA complex and enhanced formation of a basal complex evident in control cells (Ref. 12 and Fig. 6Go). Using a radiolabeled probe containing -137 to +45 bp of the 5'-flanking region of the HLA-DR{alpha} gene and FRTL-5 cell extracts from cells treated with TSH, a major upper protein/DNA complex can be seen after 48 h of autoradiography (Fig. 6AGo, lane 2). Treatment of the cells with 100 U/ml {gamma}-IFN induces or markedly increases the formation of a faster migrating or lower complex and enhances the intensity of the basal complex (Fig. 6AGo, lane 3). The same changes are evidenced by transfecting cells with pCIITA (Fig. 7AGo, lane 4), but not the pcDNA3 vector (Fig. 6AGo, lane 1). These data were duplicated in cells maintained without TSH (data not shown). Clear separation of the two complexes evident in extracts from CIITA transfected, as well as {gamma}-IFN-treated cells, was difficult to achieve (Fig. 6AGo); however, the two complexes could be readily distinguished and compared by differences in their protein composition, as evidenced using specific antisera, and by differences in oligonucleotide competition studies, both of which are demonstrated in the next experiments.



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  		Figure 6. Ability of CIITA transfection to modulate the formation of protein/DNA complexes between cell extracts and a 32P-radiolabeled probe containing the 5'-flanking region of HLA-DR{alpha}, as measured by EMSA. The probe was excised by restriction enzyme treatment of the -137 to +45-bp DR{alpha}-CAT chimera and is diagrammatically represented at the bottom of the figure. FRTL-5 rat thyroid cells were grown to near confluency in TSH and either transfected with pCIITA or pcDNA3 as described in Figs. 1 to 4GoGoGoGo or treated with 100 U/ml {gamma}-IFN. Cell extracts were prepared 48 h after transfection or the start of IFN treatment using a modification of the method of Dignam et al. (40) as described in Materials and Methods. In panel A, extracts were incubated with the 32P-radiolabeled probe containing -137 bp of the DR{alpha} 5'-flanking region and EMSA performed as described (Materials and Methods). In Panel B, the extract from cells transfected with pCIITA was preincubated with the following antisera from Santa Cruz (detailed in Materials and Methods) before being evaluated for its ability to form complexes: CREB-1, CREB-2, ATF-2, ATF-3, ATF-1, ATF-4, CBP, NF-{kappa}Bp65, and NF-{kappa}Bp50. In panel C, the extract from cells transfected with pCIITA was preincubated with a double-stranded oligonucleotide, -87 to -55 bp of the HLA-DR{alpha} 5'-flanking region, which contains the Y box element. The arrows denote the basal and IFN/CIITA-induced complexes; the autoradiograms were exposed 48 h at -70 C.

 


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  		Figure 7. Effect of transfection of the thyroid Y box protein, TSEP-1, on {gamma}-IFN- (A) and CIITA-increased (B) HLA-DR{alpha} gene expression in FRTL-5 thyroid cells. In panel A, FRTL-5 cells were grown to near confluency in medium with TSH (6H medium) and cotransfected with -137-bp HLA-DR{alpha}-CAT, pRSV-luciferase, and either 20 µg pRcCMV-TSEP-1 or its pRcCMV control vector, then treated with 100 U/ml {gamma}-IFN. In panel B, duplicate sets of cells were cotransfected with -137-bp HLA-DR{alpha}-CAT, pRSV-luciferase, and combinations of the following as noted: 15 µg pCIITA or pcDNA3 and 50 µg pRcCMV-TSEP-1 or pRcCMV. In both panels, CAT activity was measured 48 h later as described ( Figs. 1–5GoGoGoGoGo and Materials and Methods). Cell viability was approximately 80% in all samples. In Panel A, results are expressed relative to -176-bp HLA-DR{alpha}-CAT activity in the absence of {gamma}-IFN (first open bar); in panel B, results are expressed relative to -176-bp HLA-DR{alpha}-CAT activity in cells cotransfected with pRcCMV. Data in all cases were corrected both for luciferase activity and cell protein; these corrections in all cases resulted in less than 6.5% change in activity. Results are the mean ±SD of three separate experiments performed on three different batches of cells. One asterisk (*) denotes a significant increase (P < 0.01) in activity induced either by {gamma}-IFN or pCIITA cotransfection; two asterisks (**) denote a statistically significant decrease in {gamma}-IFN-or CIITA-increased DR{alpha} promoter activity induced by pRcCMV-TSEP-1.

 
Third, we evaluated the characteristics of the complexes in cells treated with {gamma}-IFN or transfected with pCIITA to determine whether they had similar properties (Ref. 12; Fig. 6Go, B and C). Exactly as described for {gamma}-IFN (12), the faster migrating complex increased by transfecting cells with pCIITA is nearly eliminated if extracts are incubated with an antiserum to CBP (Fig. 6BGo, lane 10), whereas the upper complex is markedly reduced by antisera to activating transcription factor-1, ATF-1, or ATF-2 (Fig. 6BGo, lanes 4 and 6, respectively). CBP is a coactivator of cAMP response element (CRE) binding proteins (16); ATF-1 and ATF-2 are CRE-binding proteins (CREBs) (47). The changes are specific, i.e. are not duplicated by antisera to other CREBs, CREB-1, CREB-2, ATF-3, ATF-4, or the subunits of NF-{kappa}B, p50 or p65 (Fig. 6BGo). In addition, formation of the pCIITA-induced lower complex, exactly as was the case for the {gamma}-IFN-increased lower complex (12), is specifically prevented by a double strand oligonucleotide containing the Y box sequence (Fig. 6CGo), but not the X or S boxes (data not shown), when they were included in the gel shift assay in vitro.

In sum, it is reasonable to conclude that {gamma}-IFN induces the formation of CIITA in thyrocytes and that CIITA is an important intermediate in the ability of {gamma}-IFN to increase HLA-DR{alpha} gene expression in FRTL-5 thyrocytes. Thus, in addition to causing similar, nonadditive changes in functional expression of the class II gene, its interaction with transcription factors, once its mRNA transcript is induced by {gamma}-IFN, results in the same alterations in complex formation as does {gamma}-IFN: induction or increased formation of a novel, faster migrating complex with the HLA-DR{alpha} promoter and enhanced formation of a basal complex. Moreover, the faster migrating novel complex in the pCIITA-transfected and {gamma}-IFN-treated cells is similar in its sensitivity to anti-CBP and the Y box double strand oligonucleotide, whereas the basal complex is particularly sensitive to antisera to ATF-1 and ATF-2. Although these data do not establish that all components of the complexes in extracts of the pCIITA-transfected and {gamma}-IFN-treated FRTL-5 cells are identical, they certainly support their relatedness at this point. The following experiments provide additional support of their relatedness.

The ability of either {gamma}-IFN or CIITA to increase HLA-DR{alpha} gene expression in FRTL-5 thyroid cells is inhibited by overexpression of the thyroid Y box protein, TSEP-1
Using transient transfections and CAT assays, we evaluated the effect of pTSEP-1 on the ability of {gamma}-IFN (Fig. 7AGo) or CIITA (Fig. 7BGo) to increase -176-bp DR{alpha}-CAT chimera activity in cells maintained in the presence of TSH. HLA-DR{alpha} gene expression is significantly lower in cells treated with 100 U/ml {gamma}-IFN but transfected with pRcCMV-TSEP-1 (Fig. 7AGo) or cells transfected with pRcCMV-TSEP-1 plus pCIITA (Fig. 7BGo). The effect of pRcCMV-TSEP-1 is not duplicated by cotransfection of the vector alone, pRcCMV, without the full-length TSEP-1 cDNA insert (Fig. 7Go, A and B). As little as 10 µg pRcCMV-TSEP-1 is able to nearly fully suppress (87 ± 6%) the effect of 100 U/ml {gamma}-IFN; only 2.5 µg suppresses the effect of 50 U/ml {gamma}-IFN. The amounts needed to achieve similar suppression of pCIITA (Fig. 7BGo) are much higher, i.e. 50 µg pRcCMV-TSEP-1 using 15 µg pCIITA; however, using 5 µg pCIITA, only 15 µg pRcCMV-TSEP-1 is needed for 82.5 ± 7% inhibition.

We showed earlier that CIITA induction of HLA-DR{alpha} CAT activity required not only the S, but also the X1, X2, and Y boxes. Thus, mutation of each element alone resulted in a loss in the increase effected by CIITA overexpression and a return toward levels present when the vector alone was the cotransfectant (Fig. 3Go). Residual activity exhibited by HLA-DR{alpha}-CAT constructs with mutations of the S, X1, and X2 boxes (Fig. 3Go) was eliminated by cotransfection with pRcCMV-TSEP-1 but not by cotransfection with pRcCMV, which does not contain the TSEP-1 insert (Fig. 8Go).



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  		Figure 8. Ability of TSEP-1 to decrease residual class II promoter activity after FRTL-5 thyroid cells are cotransfected with the pCIITA and a -176-bp DR{alpha}-CAT construct having mutations within the S, X1, and X2 boxes. FRTL-5 cells grown to near confluency in medium with TSH (6H medium) were cotransfected with the noted DR{alpha}-CAT chimera, pRSV-luciferase, pCIITA, and either pRcCMV-TSEP-1 or its vector control pRcCMV (Materials and Methods; Figs. 3Go or 7). CAT activity was measured 48 h later. Results are expressed relative to CAT activity of cells transfected with the same DR{alpha}-CAT chimera, pRSV-luciferase, pRcCMV-TSEP-1 or pRcCMV, and the pcDNA3 vector control rather than pCIITA, whose activity is set at unity and is evident in Fig. 7Go relative to vector controls; in all cases, CAT activities are first corrected both for luciferase activity and cell protein. These corrections resulted in less than 5% change in activity. Results are the mean ±SD of three separate experiments performed on three different batches of cells. A single asterisk (*) denotes a statistically significant decrease (P < 0.05) in DR{alpha} promoter activity induced by pRCcMV-TSEP-1; the dashed line represents the value of the controls with pcDNA3, which are set at 1. Mutations are detailed at the top of Fig. 3Go.

 
Consistent with its action to decrease HLA-DR{alpha}-CAT activity (Figs. 7Go and 8Go), TSEP-1 overexpression caused a decrease in the formation of the novel, faster-migrating protein/DNA complex present in {gamma}-IFN-treated (Fig. 9Go, lanes 3 and 4 vs. 1) or pCIITA-transfected (Fig. 9Go, lanes 6 and 8 vs. 5 or 10) FRTL-5 thyroid cells, by comparison to transfection with the pRcCMV control vector (Fig. 9Go, lanes 2 and 7, respectively), which had a negligible effect. Measured by densitometry as a function of the amount of pTSEP-1 DNA cotransfected, 10, 25, and 50 µg, the decrease was 16 ± 6%, 42 ± 6%, and 81 ± 7%, respectively.



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  		Figure 9. Ability of TSEP-1 transfection to alter the {gamma}-IFN- (lanes 1–4) or CIITA-increased (lanes 5–9) protein/DNA complexes formed between cell extracts and a 32P-radiolabeled probe containing the 5'-flanking region of HLA-DR{alpha}. The probe was the same as that used in Fig. 6Go and contains -137 to +45 bp of the 5'-flanking region of HLA-DR{alpha}. FRTL-5 rat thyroid cells were grown to near confluency in TSH, transfected with either 30 (1x) or 50 (2x) µg pRcCMV-TSEP-1 or 50 µg of its control counterpart, pRcCMV, then treated with 100 U/ml {gamma}-IFN (lanes 1–4). Alternatively, cells were cotransfected with pCIITA, pRcCMV-TSEP-1, or pRcCMV, as noted, and maintained in medium with no {gamma}-IFN (lanes 5–8 and 10). Cell extracts were prepared 48 h after transfection or the start of {gamma}-IFN treatment, as in Fig. 7Go and as described in Materials and Methods. Extracts were incubated with the 32P-radiolabeled probe containing -137 to +45 bp of the DR{alpha} 5'-flanking region and EMSA performed as in Fig. 6Go or as described in Materials and Methods. The arrows denote the basal and IFN/CIITA-induced complexes; the autoradiograms were exposed 48 h at -70 C. The EMSA pattern of a control extract from cells not treated with IFN or transfected with CIITA and autoradiographed identically is in lane 9.

 
These data indicated that the rat thyroid Y-box homolog of human YB-1, which decreases TSHR and MHC class I gene expression in FRTL-5 thyroid cells (29, 30, 31, 32), also decreases or prevents aberrant MHC class II gene expression. Further, the Y box protein counterbalances the action of CIITA just as it does {gamma}-IFN, further supporting the conclusion that CIITA is an intermediate factor in the {gamma}-IFN signal pathway in FRTL-5 thyroid cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aberrant MHC class II expression on nonimmune cells has been associated with multiple autoimmune diseases other than ATD (1, 2, 3, 48, 49, 50). The assumption emerged that aberrant class II expression allowed cells to become antigen-presenting cells, interact with T cells, and initiate an immune response (1, 2, 3, 7, 8, 9, 48, 49, 50, 51). A recent study (4) supports this hypothesis. Thus, immunizing mice with fibroblasts transfected with the human TSHR and a MHC class II molecule, but not either alone, induced ATD with the major humoral and histological features of Graves’ disease. Under these circumstances, it seemed important to examine what factors and elements control class II expression at a transcriptional level in normal thyrocytes in order to understand the basis for its normal suppressed state, as well as the basis for the loss of suppression. We used FRTL-5 thyroid cells because they have been used in studies of aberrant class II expression induced by {gamma}-IFN (7, 8, 9), because the changes induced by {gamma}-IFN mimic those in ATD (7, 8, 9, 10), and because {gamma}-IFN has been implicated as a primary or secondary factor in aberrant class II expression (5, 6, 48, 51).

In our separate studies (11, 12), we reported that the HLA-DR{alpha} promoter-CAT activity is normally not expressed in FRTL-5 thyroid cells, mimicking the absence of endogenous class II gene expression. We showed, however, that {gamma}-IFN induces HLA-DR{alpha} gene expression in FRTL-5 cells, as it does endogenous class II expression, and that the same elements, S, X1, X2, and Y-boxes, are required for {gamma}-IFN to increase MHC class II gene expression in thyrocytes, as in antigen-presenting cells of the immune system. A novel finding was that {gamma}-IFN treatment of FRTL-5 cells induced or increased the formation of an additional protein/DNA complex with the 5'-flanking region of the HLA-DR{alpha} gene, that its formation was specific and was specifically inhibited by a double strand oligonucleotide containing the Y box element. Using specific antisera, we provided evidence that the {gamma}-IFN-induced faster migrating complex contained CBP as an important component and that the {gamma}-IFN-increased basal complex involved two transcription factors associated with binding to cAMP response elements (CREs), ATF-1 and ATF-2 (12). CBP is thought to recruit CRE binding proteins and link them with basal transcription factors including RNA polymerase II and be critical for the expression of cAMP-responsive genes, growth, function, and IFN action via the JAK-STAT pathway (16). These last results were consistent with the fact that the X2 box is homologous to a CRE (5, 6, 13, 14, 15), that the X2 site is required for aberrant expression of MHC class II antigens in SV40 large T antigen-transformed COS cells (52), and that in antigen-presenting cells from the immune system the region including the S, X1, X2, and Y boxes on HLA genes forms a multimeric complex involving a multiplicity of CREBs in association with RF-X1–4, CIITA, YB-1, NF-X, NF-Y, and X2bp (5, 6, 13, 14, 15, 21, 22, 23, 24, 25).

In this report, we have evaluated the roles of CIITA and the rat thyrocyte-derived homolog of the human Y box protein, YB-1, in {gamma}-IFN action and aberrant expression of the HLA-DR{alpha} gene. CIITA is a regulatory gene mutated in cells from patients with the bare lymphocyte syndrome who do not exhibit an RFX-binding defect but who still exhibit defective class II gene expression (6, 20, 21, 22, 23, 24, 25). It was cloned using a complementation cloning strategy (20) and suggested to be a protein-protein binding factor rather than a DNA-binding factor. In lymphocytes, its presence is constitutive and is suggested to account for constitutive class II gene expression; in antigen-presenting cells from the immune system, it is regarded as a molecular switch whose presence can be induced by cytokines and therefore controls inducible MHC class II gene transcription (6, 20, 21, 22, 23, 24, 25). In this report we show that CIITA functions as an intermediate in {gamma}-IFN-induced aberrant class II gene expression in thyrocytes. Thus, it duplicates the action of {gamma}-IFN and has the same properties as that exhibited by {gamma}-IFN in terms of a functional requirement for multiple conserved elements on the class II promoter, S, X1, X2, and Y boxes, to express activity. It induces the formation of the same or a closely related protein/DNA complex as is the case for {gamma}-IFN (25) and is not additive with {gamma}-IFN in its action. We show that CIITA is normally not expressed at an mRNA level in FRTL-5 thyroid cells but is induced by {gamma}-IFN. These results make it reasonable to hypothesize that {gamma}-IFN induces the synthesis of CIITA in the thyrocyte. CIITA then interacts with transcription factors normally present and important for cAMP-induced expression of multiple genes regulated by TSH. This results in the formation of a new multimeric complex, and aberrant expression of the class II gene ensues. These data and this interpretation are consistent with data in other studies (6, 21, 22, 23, 24, 25).

YB-1 is the prototype Y box-binding protein. It was cloned using the radiolabeled Y box element of the class II promoter to screen a {lambda}gt11 expression DNA library (26). An intriguing feature was the inverse relationship of the levels of YB-1 and DRA in {gamma}-IFN-activated cell lines; this suggested that YB-1 might negatively regulate class II gene expression (26). Direct evidence of the ability of YB-1 to suppress {gamma}-IFN-induced class II gene expression was provided in glioblastoma cells (27). Initially, it was suggested to function by its ability to recruit or enhance binding to the class II S box, after its interaction with the double-stranded Y-box, the inverted CCAAT element of the MHC class II gene (53). More recently, it was additionally suggested that YB-1 binding to the class II promoter results in single-strand binding regions that prevent loading and/or function of other class II transcription factors (28). The Y box protein- binding site associated with this latter function is a CT-rich area 5' to the X-box, which has a CCTT motif in the human class II gene, as does the S box (13). It is suggested the Y box interaction can induce- or stabilize single-strand regions in the class II promoter by interacting with the CT-rich site 5' to the CRE-like X2 site (28).

During the course of studies of the negative regulation of the TSHR, we cloned a single-strand binding protein that interacted with a suppressor element that exists 5' to the CRE-like site of the TSHR minimal promoter and that regulated optimal expression of the TSHR by modulating its CRE-like constitutive enhancer (29). We found that we cloned a Y box protein, termed it TSEP-1 (TSHR suppressor element protein-1), and related it to another Y box protein, NSEP-1, which interacted with CT-rich promoter regions involved in single-strand or triple helix formation (29). TSEP-1 was shown, additionally, to interact with two other TSHR sites, not only the CT-rich area, and all sites were shown to have a conserved CCTC/T sequence. As was the case in the class II gene, it was suggested the Y box protein interaction with the TSHR could induce or stabilize single-strand regions by interacting with the CT-rich region 5' to the CRE-like site (29).

In this report, we show that TSEP-1 acts like YB-1 to suppress {gamma}-IFN-induced class II gene expression. Moreover, we present the novel result that it suppresses CIITA action, consistent with the role of CIITA as an intermediate in {gamma}-IFN action and the importance of Y box proteins in regulation of class II gene expression. This observation has multiple implications. First, it is not unreasonable to presume that the Y box protein is a normal suppressor of class II gene expression in thyrocytes under TSH control, accounting in part for the absence of class II expression in thyrocytes. Thus, we have shown that suppression activity is protein kinase A activated (29). We have preliminarily shown that {gamma}-IFN can decrease TSEP-1 RNA levels, whereas methimazole reverses this {gamma}-IFN action (30). We suggest, therefore, that {gamma}-IFN simultaneously reduces class II-suppressive action by decreasing TSEP-1 RNA levels and increases class II expression by increasing CIITA RNA levels. The net result is aberrant expression of MHC class II. Methimazole helps reverse this by reversing the effect of {gamma}-IFN on TSEP-1 (Y box) RNA levels (30) and eliminating the {gamma}-IFN-induced complex with the HLA-DR{alpha} 5'-flanking region (12).

Second, Y box proteins are not normally associated with suppression when they bind to nuclease-sensitive, CT-rich domains of genes with GC-rich promoters, but, rather, activation of growth-related genes: c-myc, the epidermal growth factor receptor, and c-Ki-ras (54, 55). Sabath et al. (56) have, in fact, suggested that TSEP-1 might stimulate the transcription of numerous growth-associated genes, i.e. the Y box protein may be an important factor in the regulation of cell growth while suppressing MHC class I and class II gene expression. Since TSEP-1 can decrease class I levels in FRTL-5 thyrocytes (30, 31, 32), as well as TSHR (29) and class II gene expression, the data support the hypothesis that common transcription factors are involved in the negative regulation of each and that this allows the cross-talk necessary to prevent class I or class II antigens from presenting TSH-increased proteins, which are a consequence of TSH-increased function or growth response, to immune cells. Self-tolerance is preserved and autoimmunity prevented.

In sum, the present results demonstrate that, in thyrocytes, CIITA mediates IFN action and connects regulation of class II expression to the same sequence elements as in antigen-presenting cells from the immune system. It demonstrates that regulation of class II expression appears to involve the counterbalanced actions of TSEP-1 and CIITA.


   Footnotes
 
1 V.M., M.S., and C.G. 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 TRAC Award from the Knoll Pharmaceutical Company (Mount Olive, NJ) during the course of this project. A. M. R. is supported by a Clinical Investigator Award from the NIH (KO8AR-01915). Back

2 Current address: First Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan. Back

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

4 Current address: Third Department of Internal Medicine, Yamanashi Medical University, 1110 Tamaho-cho, Nakakomagun, Yamanashi-ken, 409–38 Japan. Back

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

Received April 3, 1997.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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