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Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (M.S., S.-i.T., K.S., C.G., G.N., J.S., M.S., B.F., L.D.K.), and Experimental Immunology Branch, National Cancer Institute (D.S.S.), National Institutes of Health, Bethesda, Maryland 20892; the Department of Surgery, Johns Hopkins University (M.S.), Baltimore, Maryland 21287; and the Departments of Cancer Biology and Medicine, Harvard School of Public Health and Harvard Medical School (A.M.R.), Boston, Massachusetts 02115
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN). We have studied IFN-induced human leukocyte
antigen (HLA)-DR
gene expression in rat FRTL-5 thyroid cells to
identify the elements and factors important for aberrant expression.
Using an HLA-DR 5'-flanking region construct from -176 to +45 bp
coupled to the chloramphenicol acetyltransferase reporter gene, we show
that there is no basal class II gene expression in FRTL-5 thyroid
cells, that IFN can induce expression, and, as is the case for
antigen-presenting cells from the immune system, that IFN-induced
expression requires several highly conserved elements on the
5'-flanking region, which, from 5' to 3', are the S, X1,
X2, and Y boxes. Methimazole (MMI), a drug used to treat
patients with Graves’ disease and experimental thyroiditis in rats or
mice, can suppress the IFN-induced increase in HLA-DR gene
expression as a function of time and concentration; MMI simultaneously
decreases IFN-induced endogenous antigen presentation by the cell.
Using gel shift assays and the HLA-DR 5'-flanking region from -176
or -137 to +45 bp as radiolabeled probes, we observed the formation of
a major protein-DNA complex with extracts from FRTL-5 cells untreated
with IFN, termed the basal or constitutive complex, and formation of
an additional complex with a slightly faster mobility in extracts from
cells treated with IFN. MMI treatment of cells prevents IFN from
increasing the formation of this faster migrating complex. Formation of
both complexes is specific, as evidenced in competition studies with
unlabeled fragments between -137 and -38 bp from the start of
transcription; nevertheless, they can be distinguished in such studies.
Thus, high concentrations of double stranded oligonucleotides
containing the sequence of the Y box, but not S, X1, or
X2 box sequences, can prevent formation of the
IFN-increased faster migrating complex, but not the basal complex.
Both complexes involve multiple proteins and can be distinguished by
differences in their protein composition. Thus, using specific
antisera, we show that two cAMP response element-binding proteins,
activating transcription factor-1 and/or -2, are dominant proteins in
the upper or basal complex. The upper or basal complex also includes
c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes
the IFN-increased lower complex is CREB-binding protein (CBP), a
coactivator of cAMP response element-binding proteins. We, therefore,
show that aberrant expression of MHC class II in thyrocytes, induced by
IFN, is associated with the induction or increased formation of a
novel protein-DNA complex and that its formation as well as aberrant
class II expression are suppressed by MMI, a drug used to treat human
and experimental autoimmune thyroid disease. Its component proteins
differ from those in a major, basal, or constitutive protein-DNA
complex formed with the class II 5'-flanking region in cells that are
not treated with IFN and that do not express the class II gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One form of ATD is Graves’ disease, in which autoantibodies develop to the TSH receptor (TSHR) and induce hyperthyroidism by mimicking the action of TSH. Whereas numerous attempts to develop a Graves’ disease model by immunizing animals with the extracellular domain of the TSHR have largely failed (7, 8, 9, 10, 11, 12, 13, 14, 15, 16), immunizing mice with fibroblasts transfected with the human TSHR and a MHC class II molecule, but not either alone, has induced ATD with the major humoral and histological features of Graves’ disease (17): stimulating TSHR antibodies, TSH binding-inhibiting Igs that are different from stimulating TSHR antibodies, increased thyroid hormone levels, thyroid enlargement, and thyrocyte hypercellularity. These results support the hypothesis that aberrant expression of MHC class II molecules and acquisition of antigen-presenting ability on a thyroid cell may activate T and B cells normally present in an animal, thereby allowing normal immune tolerance to be broken. Understanding the basis for aberrant class II expression in thyrocytes may, therefore, be a potentially important aspect of understanding the pathogenesis of ATD and Graves’ disease.
Interferon- (IFN) is the most potent inducer of class II gene
expression; its ability to induce class II expression is well studied
in antigen-presenting cells of the immune system (1, 2, 3, 18, 19). In
those cells, its action is transcriptionally mediated and involves
conserved sequences present in all class II MHC promoters characterized
to date (1, 2, 3, 18, 19). From 5' to 3', these are the S (also called W
or H), X1, X2, and Y boxes (Fig. 1
). Despite the fact that IFN can
induce aberrant class II antigen expression when given in
vitro to human (20) or rat FRTL-5 thyroid cells in culture
(21, 22, 23) and can mimic the autoimmune state of thyrocytes in ATD
(4, 5, 6), little is known about the transcriptional mechanism by which
IFN modulates class II genes in thyrocytes. We, therefore, initiated
a study of HLA-DR gene expression in rat FRTL-5 thyroid cells to
define elements and factors important for IFN-induced aberrant
expression in thyrocytes and, by inference, ATD. We additionally
evaluated the effect of methimazole (MMI) on IFN-induced class II
expression, as it is an agent used to treat Graves’ disease (24) and
other forms of autoimmunity (25, 26, 27) and is capable of suppressing MHC
class I gene expression in FRTL-5 thyroid cells (28).
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-increased MHC class II
expression in FRTL-5 thyrocytes, like antigen-presenting cells from the
immune system, requires the highly conserved 5'-elements present in all
MHC class II promoters: the S, X1, X2, and Y
boxes. We show that MMI can inhibit the action of IFN and identify a
novel protein-DNA complex whose formation is induced or enhanced by
IFN and suppressed by MMI in association with their opposite effects
on class II gene expression. We hypothesize that formation of this
protein-class II promoter complex in thyroid cells may be associated
with aberrant class II expression in ATD and that suppression of its
formation may be a component of the action of MMI in ATD. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were
purchased from Life Technologies (Grand Island, NY)m and rabbit
polyclonal antibodies against the components of the activating
transcription factor (ATF)/cAMP response element-binding protein
(CREB), Fos/Jun, nuclear factor-
B (NF-B), Stat, IFN response
element (IRF), Ets, and Oct families of proteins were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA).
[-32P]Deoxy-CTP (3000 Ci/mmol) and
[14C]chloramphenicol (50 mCi/mmol) were purchased from
DuPont-New England Nuclear (Boston, MA); [-32P]ATP
(6000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL).
Synthetic oligonucleotides were purchased from Operon Technologies
(Alameda, CA). The source of other materials was Sigma Chemical Co.
unless otherwise noted.
Plasmids
HLA-DR promoter constructs 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 were previously described (30). An additional
chloramphenicol acetyltransferase (CAT) plasmid containing -38 to +45
bp of the 5'-flanking region of HLA-DR was constructed by PCR, using
the HLA-DR-CAT chimera containing -176 to +45 of 5'-flanking region
as template and the following primers: a 5'-primer with a
5'-SphI restriction site,
5'-ACATGCATGCGGTCAGACTCTATTACACCCCAC-3', and a 3'-primer,
5'-CTAGTCTAGTTTGGGAGTCAGTAGAGCTCG-3', with an XbaI
restriction site (30). The PCR products were purified by
phenol-chloroform extraction, digested with XbaI and
SphI, purified from a 2% agarose gel with Jet Sorb
(Genomed, Frederick, MD), dephosphorylated with calf intestinal
alkaline phosphatase (New England Biolabs, Beverly, MA), and religated
(DNA ligation kit, Takara Biochemical, Berkeley, CA), into the
pCAT-Basic Vector (Promega, Madison, WI) at the SphI
and XbaI sites as previously described (30).
The thyroglobulin (TG)-CAT construct, pTG-688-CAT, was derived by
substituting the TG promoter insert from our previously described
pTG-CAT chimera (31) into the HLA-DR-CAT chimera from which the
class II promoter insert was excised. The vector containing the CAT
reporter gene but no insert was the control.
DNA was prepared and purified by CsCl gradient centrifugation (32). The sequences of all constructs were confirmed by a standard method (33).
Cell culture
FRTL-5 rat thyroid cells (Interthyr Research Foundation,
Baltimore, MD; American Type Culture Collection CRL8305) were a fresh
subclone (F1) with the properties previously described (28, 34, 35).
They were grown in Coon’s Modified F-12 Medium containing 5%
heat-treated, mycoplasma-free calf serum, 1 mM nonessential
amino acids, 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 5th
and 25th passage. Fresh medium was added every 2 or 3 days, and cells
were passaged every 7–10 days. In some experiments, cells were grown
to near confluence in 6H medium, then maintained for 6–8 days, before
experiments were initiated, in 5H medium with no TSH.
Transient expression analysis
Transient transfections in FRTL-5 cells were performed as
previously described (36, 37), using one of the following procedures.
In one, cells were cultivated in 6H medium to approximately 80%
confluence, 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 CAT chimera together with 2 µg pRSV-luciferase) (38), which
was used to measure the efficiency of transfection. Cells were pulsed
(330 V; capacitance, 25 µfarad), plated (6 x 106
cells/10-cm dish), and cultured in 6H medium plus 5% calf serum
supplemented or not with 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% confluence in 6H medium,
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 IFN. Cell viability was
approximately 80% in all experiments. CAT activity was measured as
previously described (36, 37); values were normalized to luciferase
activity measured using the Promega (Madison, WI) assay system.
Cellular extracts
Cell extracts were made by a modification of the method of
Dignam et al. (39). 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 Dignam buffer C [20 mM
HEPES (pH 7.9), 1.5 mM MgCl2, 0.42
M NaCl, 25% glycerol, 0.5 mM dithiothreitol,
0.5 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin,
and 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 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) or were purified from 2% agarose gel using either QIAEX
(Qiagen, Chatsworth, CA) or Jet Sorb (Genomed) after restriction enzyme
treatment of the chimeric CAT constructs described above. They were
dephosphorylated with calf intestinal alkaline phosphatase, labeled
with [-32P]deoxy-CTP using Klenow or with
[-32P]ATP using T4 polynucleotide kinase, then
purified on an 8% native polyacrylamide gel (36, 37, 40, 41, 42).
EMSAs were performed basically as previously described (36, 37, 41, 42). Binding reactions were carried out in a volume of 20 µl for 30 min at room temperature. The reaction mixtures contained 1.5 fmol [32P]DNA, 3 µg cell extract, and 1.5 µg poly(deoxyinosinic-deoxycytidylic) in 10 mM Tris-Cl (pH 7.9), 1 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol. Where indicated, unlabeled double or single stranded oligonucleotides were added to the binding reaction as competitors and incubated with the extract for 20 min at room temperature before the addition of labeled DNA. Similarly, in experiments using antiserum, extracts were incubated in the same buffer containing antiserum or normal rabbit serum for 20 min at room temperature before being processed as described above. After incubations, reaction mixes were subjected to electrophoresis on 3.5% or 5% native polyacrylamide gels at 160 V in 0.5 x Tris-borate-EDTA at room temperature for 1.5–2 h. Gels were dried and autoradiographed at -80 C overnight unless otherwise noted.
Other methods
The protein concentration was determined by Bradford’s method
(Bio-Rad, Richmond, CA); recrystallized BSA was the standard.
For fluorescence-activated cell sorter analysis, single cell suspensions were prepared and stained as previously described (17, 43), except that HLA-DR antigen was detected using a class II-specific murine monoclonal antibody (44); Leu-4 was used as a background control.
Statistical significance
All experiments were repeated at least three times with
different batches of cells. Values are the mean ± SE
of these experiments as noted. Significance between experimental values
was determined by two-way ANOVA and were significant at
P < 0.05 when data from all experiments were
considered.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in FRTL-5 thyroid cells requires IFN and
the same 5'-promoter elements important for IFN induction of
HLA-DR in lymphocytes -176 bp minimal promoter coupled to CAT, as
previously described and used in studies of immune cells (30), to
evaluate expression of the HLA class II gene in FRTL-5 thyrocytes.
HLA-DR is not expressed in transiently transfected FRTL-5
thyrocytes, compared to the vector control, unless the cells are
treated with rat recombinant IFN (Fig. 2A, first and second sets of open
and closed bars). The action of rat IFN is not duplicated by
human IFN and is associated with an increase in endogenous class II
expression measured by flow cytometry (see below, Fig. 8),
i.e. its action is specific and appears to reflect effects
on the endogenous class II antigen.
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-CAT
chimera to -137, -122, -111, -97, and -38 bp showed that, like
immune cells (2, 18, 19), IFN induction is lost once the S box,
-137 to -123 bp, is removed (Fig. 2A
). Also, like immune cells (2, 18, 19), IFN induction requires not only the S box, but also the
X1, X2, and Y boxes, for activity. Thus,
mutation of each element individually also resulted in the loss (Fig. 2B, MUT S, MUT X1, and MUT Y) or a significant decrease
(Fig. 2B, MUT X2) in the IFN response.
In summary, unlike lymphocytes (2, 18, 19, 30), there is no
constitutive or basal expression of the HLA-DR promoter-CAT chimera
in thyrocytes. Like monocytes, however (2, 18, 19), IFN increases
HLA-DR promoter expression and requires the same highly conserved
5'-flanking region elements, S, X1, X2, and Y,
that are present in all class II genes for this effect.
The effect of TSH on IFN-induced HLA-DR gene expression in
FRTL-5 thyroid cells
TSH has been reported to enhance the ability of IFN to increase
class II antigen expression in rat FRTL-5 thyroid cells when measured
after 48 h of IFN treatment (21). We, therefore, evaluated the
effect of IFN on DR gene expression in FRTL-5 thyroid cells
maintained with and without TSH, as this might have influenced
subsequent experiments.
In the presence of TSH, the effect of IFN on class II gene
expression was measurable within 12 h and maximal at 48 h
(Fig. 3A); in the absence of TSH, it was
also measurable by 12 h, was maximal at 24 h, and decreased
slightly, but significantly, by 48 h (Fig. 3B). Consistent with
these data, IFN-induced endogenous class II antigen expression,
measured by flow cytometry (data not shown), was higher at 48 h in
FRTL-5 thyroid cells transfected with the HLA-DR-CAT chimera and
maintained with TSH than in cells maintained without TSH. The present
data are thus consistent with and might explain why IFN-increased
antigen expression was higher after 48 h of treatment in the
presence of TSH (21), i.e. TSH delays the ability of IFN
to maximally increase gene expression. Both studies suggest that the
effect of TSH on IFN activity is a kinetic phenomenon.
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-induced HLA-DR gene expression in
FRTL-5 thyroid cells to increase class II gene expression
was dependent on its concentration regardless of whether the -176 bp
(data not shown) or -137 bp (Fig. 4A)
DR-CAT chimera was used in transient transfection assays. The
maximal effect was in each case evident at 100 U/ml IFN (Fig. 4A).
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to increase class II
promoter activity in the FRTL-5 thyrocytes.
MMI prevents the ability of a maximally effective concentration
of IFN to increase the activity of the -137 bp DR-CAT chimera as
a function of time. Thus, CAT activity induced by 100 U/ml IFN was
progressively decreased 24 and 48 h after MMI addition (Fig. 4B).
The MMI concentration in this experiment (5 mM) was
previously shown to be maximally effective to suppress IFN-induced
MHC class I expression in these cells (28); however, the effect was
evident at lower MMI concentrations and was dependent on the
concentration of MMI (Fig. 5). MMI had no
effect on promoter in the absence of IFN (Fig. 5). The actions of
both IFN and MMI were specific; thus, neither affected the control
vector, and each had opposite effects on a TG-CAT chimera (Table 1): IFN decreased TG-CAT activity, and
MMI reversed the IFN-induced decrease in TG-CAT activity. Also, MMI
alone increased TG-CAT activity, but not HLA-DR CAT activity (Table 1).
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treatment of FRTL-5 thyrocytes induces the formation of a
specific protein/DNA complex with the DR 5'-flanking region; MMI
inhibits its formation 5'-flanking
region as the radiolabeled probe, we found that a single major
protein-DNA complex appeared to form with extracts from FRTL-5 cells
maintained in the absence or presence of TSH (Fig. 6, lanes 2 and 4, respectively), although
the complex was slightly enhanced in intensity in the TSH-treated cells
(Fig. 6, lanes 4 vs. 2). In cells treated with 100 U/ml
IFN, which maximally increases CAT activity, an additional faster
migrating complex became visible, whose separate nature or existence
was more evident in TSH-treated cells (Fig. 6, lanes 3 vs. 2
and lanes 5 vs. 4), and there was an increase in the
intensity of the major complex evident in cells maintained without
IFN (Fig. 6, lanes 3 vs. 2 and lanes 5 vs. 4).
Clear separation of the two complexes was difficult to achieve;
however, they could be readily distinguished by their different
sensitivities to MMI, differences in their protein composition, and
differences in oligonucleotide competition studies, as will be
demonstrated below.
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-induced aberrant
HLA-DR expression, treatment of cells with 5 mM MMI
suppressed the ability of IFN to increase the formation of the
faster migrating complex. This is illustrated for cells treated with
IFN in the absence of TSH (Fig. 7, lanes 2 vs. 1); however, the same data were obtained in
cells treated with IFN in the presence of TSH. These data were
independent of the probe used: -137 HLA-DR or -176 HLA-DR. MMI
did not eliminate the major or basal complex formed in extracts from
cells maintained with or without IFN (Fig. 7, lanes 3 and 5
vs. 4, respectively), although it did slightly decrease its
formation (Fig. 7, lanes 5 vs. 4) just as IFN increased
its formation (Fig. 6). Consistent and coincident with its effect on
IFN-induced complex formation (Fig. 7) and IFN-induced exogenous
class II gene expression in CAT assays (Figs. 4 and 5), MMI decreased
endogenous class II antigen expression on the cell surface, as
determined by flow cytometry (Fig. 8).
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was specific. Thus,
both complexes were prevented from forming by including unlabeled -137
HLA-DR (Fig. 9, lane 3) in the
incubation in a self-competition experiment. Self-competition was
concentration dependent (34% at a 50-fold excess of unlabeled -137
HLA-DR, 69% at a 75-fold excess, and 100% at a 100-fold excess in
a representative experiment.
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, lane 3) but not a 100-fold excess of a -38 to +45
bp fragment (Fig. 9B, lane 6). Although residual complexes with
different mobilities could be detected, formation of the complexes was
still largely prevented in incubations with the -137 bp HLA-DR
fragment containing mutations of a single element, the S,
X1, X2, or Y boxes (Fig. 9B, lanes 2–5,
respectively). In studies with lymphocytes (30), these data suggested
that binding of complexes with this region of the HLA-DR 5'-flanking
region involved more than one conserved site and/or sites other than
the four conserved elements. However, we noted that a 100-fold excess
of the -90 HLA-DR fragment containing only the Y box site could
completely inhibit the formation of both complexes (Fig. 9A, lane 5) as
well as a 100-fold excess of the -137 and -111 bp fragments (Fig. 9A, lanes 3 and 4). This suggested that the Y box plus 3'-sequences to -38
bp were particularly important in complex formation, but the presence
of the other conserved elements, S, X1, and X2,
might overcome its mutation to account for the data in Fig. 9B.
Supporting the importance of the Y box as a binding site, at
least for the IFN-induced lower complex, and distinguishing it from
the basal or constitutive complex were competition studies using small
oligonucleotide fragments comprising the sequences of the S,
X1, X2, or Y boxes (Fig. 10). Thus, formation of the
IFN-induced lower complex was specifically prevented by a 200-fold
excess of a smaller, double strand oligonucleotide, -87 to -55 bp,
containing the Y box sequence (Fig. 10, bottom), but not a
200-fold excess of an oligonucleotide with the sequence of the
X1-X2 or S boxes, when each was included in the
gel shift assay in vitro (Fig. 10, lanes 7 vs.
3–6 and 8–11). A 500-fold excess of the X1-X2
or S boxes, when each was included in the gel shift assay in
vitro, was also unable to prevent formation of either complex
(data not shown). Also unable to inhibit formation were 200-fold (Fig. 10) or 500-fold (data not shown) excesses of the sense or antisense
single strand oligonucleotides with the Y,
X1-X2, or S box sequences of the minimal
HLA-DR promoter (Fig. 10 and data not shown). Inhibition was not
duplicated by single or double strand oligonucleotides with the
sequence of the S plus X1 boxes, -141 to -92 bp (data not
shown), or with the sequence of the HLA-DR promoter between -182 to
-144 bp, which served as a negative control (Fig. 10, lanes 12–14).
Thus, the Y box element is particularly important in the formation of
the IFN-induced complex (Fig. 9A, lane 5, and Fig. 10, lane 7),
whereas the Y box element plus the 3'-sequence to -38 bp are
particularly important in the formation of the basal or constitutive
complex (Fig. 9A, lane 5). This may be explained by the existence of
different protein components in each complex, as will be shown
below.
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gene in the absence of any
significant expression of the class II gene. Treatment of cells with
IFN under conditions that result in maximal class II gene expression
induces or increases the formation of a faster migrating complex, and
MMI suppresses its formation, coincident with its ability to suppress
the IFN-induced increase in class II gene expression and cell
surface antigen expression. Formation of the lower complex and
IFN-induced aberrant expression of the class II gene are, therefore,
highly correlated, whereas formation of the basal or constitutive upper
complex and its residual existence in the presence of MMI are
associated with the absence or loss of aberrant class II expression in
the FRTL-5 thyrocyte.
Characterization of proteins in the protein-DNA complexes formed
with extracts from FRTL-5 thyroid cells treated with or without
IFN
Although we observed that formation of the two complexes was still
markedly attenuated in competition experiments comparing the ability of
unlabeled oligonucleotides with mutations in each of the S,
X1, X2, and Y boxes to inhibit complex
formation (Fig. 9B), competition by an oligonucleotide with a single
site mutated resulted in small residual complexes, each with a
different mobility in the region of the upper complex (Fig. 9B, lanes
2–5 vs. 1). This suggested there might be multiple protein
components in the constitutive complex, consistent with studies in
antigen-presenting cells from the immune system (1, 2, 3), as will be
noted in Discussion. Additionally, differences in the
ability of the oligonucleotides containing the Y box or the Y box plus
the 3'-sequence to -38 bp (Figs. 10 and 9A) to compete for the
IFN-increased lower complex vs. the basal, upper complex,
respectively, suggested that each might involve different proteins. We
pursued this question using antisera against specific transcription
factors or coregulators.
An antiserum specific for ATF-1 eliminated formation of the upper,
basal, or constitutive complex and reduced the intensity of the
IFN-increased lower complex, both of which are evident in
incubations of extracts from FRTL-5 cells treated with interferon and
the DR probe containing -137 bp of the 5'-flanking region (Fig. 11, lane 6). A specific antiserum to
ATF-2 appeared to have a lesser effect on the upper complex but a
slightly greater ability to decrease the intensity of the lower complex
(Fig. 11, lanes 4 vs. 6) when analyzed quantitatively after
densitometry; however, the significance of this was unclear.
Nevertheless, this was not the case for antisera to ATF-3, ATF-4, or
two cAMP response element-binding proteins, CREB-1 or CREB-2 (Fig. 11, lanes 5, 7, 2, and 3, respectively), suggesting that these were
specific effects of the antisera, and these were specific components of
the upper or basal complex in particular. In the case of all negative
antisera, here and discussed below, the efficacy of the antiserum was
validated in control experiments showing inhibition and/or shifts using
radiolabeled oligonucleotides with consensus sequences for each
factor.
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-increased lower complex nearly completely, but did not
eliminate formation of the upper, basal complex (Fig. 11, lane 11).
Further, the same antiserum to CBP had no effect on formation of the
basal complex in extracts from cells not treated with IFN (data not
shown). Antisera against the subunits of NF-B, Stat-1, c-Jun,
activating protein-1 (AP-1), Jun-B, or Jun-D had no effect on complex
formation (Fig. 11, lanes 12–21), nor did antisera to factors
interacting with IRF-1 and IRF-2 (Fig. 11, lanes 8 and 9). These data
(Fig. 11) indicated that the two complexes differed in their protein
components and could be distinguished by antisera to ATF-1, ATF-2, and
CBP; the full extent of the difference remains to be evaluated,
however, as will be discussed below.
Several antisera were effective in altering the formation of the upper
or basal complex, as evidenced in studies using extracts from control
cells (Fig. 12). Thus, an antiserum
against c-Fos caused a shift in the migration of the complex (Fig. 12, lane 5), and antisera directed toward Fra-2, Oct-1, and Ets-2 all
inhibited formation of the complex and resulted in complexes with
slightly slower or faster migrations when added to incubations
containing extracts from cells that had not been exposed to IFN
(Fig. 12, lanes 5–8 vs. 2). Antisera against Fra-1 (Santa
Cruz preparations sc-605 or sc-183), Ets-1 (Santa Cruz preparations
sc-111 or sc-350), or Oct-2 (Santa Cruz preparation sc-233) had no
effect on either of the complexes using either extract (data not
shown), indicating specificity. The same results were obtained using
extracts from cells treated with IFN, i.e. there was no
clear indication that the lower IFN-increased complex was altered by
these antisera. However, it is evident that the migration of some of
the new complexes will overlap the mobility of the IFN-increased
lower complex (Fig. 12, lane 1 vs. 5–8). We included the Y
box double strand oligo as a competitor to eliminate any lower complex
and better track the mobility shifts in this experiment (Fig. 12, lane
4).
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fragment, but not the Y box oligonucleotide alone, to
inhibit formation of the basal, upper complex. The IFN-increased
lower complex involves CBP, but not the upper, basal complex present in
extracts from control cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which
were produced by lymphocytes infiltrating the tissue (58). More
importantly, there was little direct evidence that class II was
critical in the initiation of autoimmune thyroid disease (58). In a
recent study, however, the hypothesis became more attractive (17).
Thus, immunization of mice with fibroblasts transfected with the human
TSHR and a MHC class II molecule, but not with either alone, induced
ATD with the major humoral and histological features of Graves’
disease (17). These data established that aberrant expression of MHC
class II molecules on cells expressing the TSHR could induce ATD and
that acquisition of antigen-presenting ability as a result of aberrant
class II expression can activate T and B cells normally present in an
animal, thereby allowing normal immune tolerance to be broken. The role of MMI as an immunosuppressant able to decrease MHC class II levels on thyrocytes can also be construed as controversial. Thus, three reports did not find that MMI could decrease MHC class II antigen expression in thyrocytes (44, 59, 60), whereas a fourth (61) did. The positive study (61) used a continuously cultured, functioning, human thyroid-hybridoma cell line. The negative studies (44, 59, 60) used human thyrocytes from normal individuals and/or Graves’ patients; however, cells were maintained in high concentrations of FCS for a number of days before testing, a condition known to not only alter or eliminate some thyroid functional responses, i.e. iodide uptake, but also alter thyroid gene expression, as evidenced, for example, in studies of dog thyrocytes by the Dumont group. The possible importance of this, particularly with respect to MHC class II, is evident in one of the negative reports (44). Thus, this report showed that iodide suppressed MHC class II in FRTL-5 thyroid cells, but not in human thyrocytes. As it has recently been shown that iodide can decrease class II in the thyroids of patients being prepared for surgery and in cultured normal human thyroid cells maintained in a functional state (62), it is a reasonable possibility that there might have been a response problem in the human thyroid cells in the three negative studies.
Under these circumstances, it seemed important to examine the control
of class II expression in normal, functioning thyrocytes to understand
the mechanisms underlying aberrant class II expression, as it was a
potentially important aspect of understanding the pathogenesis of ATD.
It also seemed reasonable to see what actions MMI might have on class
II expression in thyrocytes where MMI was effective in suppressing
class I gene expression (28) and where iodide could suppress class II
and class I expression (28, 44). We thus used FRTL-5 thyroid cells as
our thyroid culture system, as it has been used in MMI and class I
promoter studies (28, 36, 37, 52, 53) as well as class II studies
(21, 22, 23, 44). We used the human HLA-DR promoter, as it has been
extensively studied in lymphocytes (2, 18, 19, 30), because regulation
of all class II genes is usually coordinate (2, 18, 19, 30), and
regulation of class II levels occurs primarily at the level of gene
transcription (2, 18, 19, 30). We used IFN as an inducer of class II
gene expression in FRTL-5 cells, as this, too, is well described
(21, 22, 23) and mimics the ability of IFN to cause aberrant class II or
increased class I expression on human thyrocytes (20, 62). Regardless
of whether IFN is a primary factor in autoimmune disease expression,
its role as an amplifier of the autoimmune response in ATD seemed clear
in studies suggesting a "vicious cycle" among T cells, monocytes,
and Graves’ or normal thyrocytes (63, 64, 65).
In the present report we establish that the HLA-DR
promoter-CAT construct is normally not expressed, as is the case for
the endogenous class II gene, in FRTL-5 (21, 22, 23) or human thyrocytes
(4, 5, 6, 20, 44, 59, 60, 61, 62). We show, however, that IFN induces HLA-DR
gene expression, as it does endogenous class II expression in FRTL-5
(21, 22, 23) or human thyrocytes (5, 6, 20, 44, 59, 60, 61, 62), and that the same
elements, S, X1, X2, and Y boxes, are required
for IFN to increase MHC class II gene expression in thyrocytes as in
antigen-presenting cells from the immune system (2, 18, 19, 30). A
novel finding is that IFN increases the formation of a specific,
multicomponent, protein-DNA complex that is different from the basal,
constitutive complex formed with the 5'-flanking region in the absence
of aberrant class II gene expression. Thus, it is distinguished from
the basal complex by the dominance of CBP as a component of the
complex, by a particularly strong or direct interaction with the double
strand Y box element of the class II promoter, as evidenced in
oligonucleotide competition experiments, and by the profound effect of
MMI to prevent IFN from inducing or enhancing its formation. MMI
only attenuates the formation of the basal or constitutive complex.
In summary, we provide reasonable evidence that MMI can suppress aberrant MHC class II gene expression in thyrocytes as well as class I expression (28) and strengthen the supposition that MMI acts as an immunosuppressant in functioning thyrocytes. The clinical relevance of aberrant class II and abnormal class I expression in Graves’ disease as well as the importance of suppressing both MHC genes to achieve clinical remission have recently been supported by the observation that iodide suppresses both class II and class I expression, which are elevated in Graves’ thyroids (62). Iodide is an agent that, like MMI, can be used to treat Graves’ patients, albeit transiently, and has been used extensively to prepare Graves’ patients for thyroidectomies.
The present report has other novel findings relative to understanding
the basis for aberrant class II expression in thyrocytes and the
ability of IFN to regulate growth and differentiation in FRTL-5
cells (21, 22, 23). Thus, it has been reported that the CRE binding motif
of the X2 site is required for aberrant expression of MHC
class II antigens in simian virus 40 large T antigen-transformed COS
cells (66), as point mutations in the X2 box decrease
transcription 20-fold. Evidence also exists that multiple CREBs can
interact with the CRE-like X2 site (67). The present data in thyrocytes
are consistent with this, in that they demonstrate that ATF-1 and/or
ATF-2, two CREBs, are components of the complexes. The novel finding,
however, is the demonstration that CBP is a dominant component of the
faster migrating IFN-increased complex and possibly contributes to
the IFN-induced increase in the intensity of the upper, basal
complex. CBP is a critical coactivator of binding proteins that bind to
the CRE, links CREBs with basal transcription factors, and is
critically involved in the regulation of cell growth and
differentiation (54, 55, 68). Signal transduction by the JAK-STAT
pathway and its regulation by IFN also involve CBP (68).
Work in human B cells identified an X box binding protein, hXBP-1, and
showed that it could bind to the 3'-end of the x box and X-Y
interspace, that it was a leucine zipper class protein with structural
similarities to the c-fos and c-jun
protooncogenes, that it formed a heterodimer with c-Fos, and that it
was important for basal class II expression in Raji cells (69, 70).
This protein regulates HLA-DR, but not HLA-DQß, gene expression
(70), i.e. it may exhibit gene and tissue specificity. It is
not yet known whether this protein is present in FRTL-5 thyroid cells;
nevertheless, this observation would be consistent with our
demonstration of c-Fos in the basal or constitutive complex. As noted
earlier, a downstream octamer sequence in HLA-DR (2, 18, 19) may
explain the Oct-1 interaction and its involvement in the basal
complex.
In a separate study (71), we show that CIITA, a protein-protein
binding factor rather than a transcription factor (67), is a mediator
of the effect of IFN to cause aberrant class II expression in FRTL-5
cells. We also show that a Y box protein, which can act as a negative
regulator of TSHR and MHC class I regulation in FRTL-5 thyroid cells
(72, 73), is a regulatory factor of class II expression and the
IFN-increased protein-DNA complex. Human YB-1 can down-regulate
class II gene expression in human glioblastoma cells (74, 75). These
last data support our hypothesis that coordinate suppression of MHC
class I and class II genes as well as the TSHR reflects the involvement
of common regulatory elements and trans factors in their
expression (52).
In summary, we have shown that IFN appears to have select
effects regulating the interaction of specific transcription factors
with the HLA-DR 5'-flanking region in FRTL-5 cells and results in a
novel, hitherto undescribed, complex with the class II gene. We show
that MMI can suppress IFN-induced aberrant class II expression and
that this is associated with its ability to inhibit the formation of a
specific and novel IFN-increased complex with the class II gene. As
a first approximation, we have been able to identify some of the
transcription factors involved in the multimeric basal or constitutive
complex formed with the HLA-DR 5'-flanking region in FRTL-5 thyroid
cells that do not express the class II gene as well as a novel
component protein in the IFN-increased complex.
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Footnotes |
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2 Current address: Department of Internal Medicine, Chungnam National
University Hospital, 640 Daesa-Dong, Chung-ku, Daejon 301–040,
Korea.
3 Current address: First Department of Internal Medicine, Tottori
University School of Medicine, Yonago 683, Japan.
4 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.
Received August 7, 1997.
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B. J Biol Chem 270:11453–11462/ß interferon treatment. Immunogenetics 30:18–26[CrossRef][Medline]
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