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-Chain in Patients with Systemic Lupus Erythematosus1
* Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910;
Department of Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20814; and
Department of Medicine, Washington Hospital Center, Washington, DC, 20005
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain.
Elf-1 (E-74-like factor) is a member of the Ets (E-26-specific) family
and is crucial for the basal transcription of TCR -chain in Jurkat
cells. We previously demonstrated that Elf-1 exists in the cytoplasm
mainly as 80-kDa form and after phosphorylation and
O-glycosylation it moves to the nucleus as a 98-kDa
which binds DNA. We now demonstrate that Elf-1 is crucial for the
transactivation of TCR -chain promoter in normal and SLE T cells.
Defective expression of TCR -chain in SLE T cells is associated with
two distinct molecular defects in the generation of the 98-kDa DNA
binding Elf-1 form. In the first, the levels of the 98-kDa form were
either decreased or absent. In the second, the apparent levels of the
nuclear Elf-1 form were normal but included only two of the three bands
into which the nuclear Elf-1 form separated in isoelectric focusing
gels. Because both the transcription and the translation processes of
Elf-1 gene are normal in SLE T cells, our data
demonstrate that abnormal posttranslational mechanisms of the Elf-1
protein result in defective expression of functional Elf-1, and
consequently, the transcriptional defect of TCR -chain in patients
of SLE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain protein and mRNA
(2). TCR -chain is a component of the CD3 complex and
is unique in that it contains more (three) phosphorylation motifs than
other CD3 components, and thus is considered critical in relaying
extracellular signals inwardly (3). This defect was
further found to correlate with the abnormal phosphorylation of the
signaling proteins (2), enhanced and sustained
Ca2+ concentration (4), and
consequent aberrant transcription of downstream genes observed in the T
cells from patients with SLE (1). Interestingly, defective
expression of the TCR -chain protein rather than mRNA has been
observed in T cells from patients with systemic infections
(5), melanoma (6), and gastric tumor
(7). In the latter, increased caspase activity has been
shown to degrade TCR -chain (8).
The transcription of TCR -chain gene has been studied in
JurkatT cells and it has been found to depend heavily on the
presence of the transcription factor Elf-1. Mutation of both Elf-1
sites on the TCR -chain promoter decreases significantly the
activity of TCR -chain promoter (9). Elf-1 is a member
of Ets family transcription factors which also includes factors such as
Ets-1, Ets-2, and Fli-1. Members of this family of transcription
factors are actively involved in the transcriptional regulation of
genes such as CD4 (10), IL-2R 
-chain (11),
and GM-CSF (12). Elf-1 gene encodes an open
reading frame of 619 aa, which should correspond to a calculated
molecular mass of 68 kDa. However, we found recently that Elf-1
exists mainly as an 80-kDa form in the cytosolic fraction of human T
lymphocytes, while in the nuclear fraction a 98-kDa Elf-1 is the
prevalent form. Phosphorylation and O-linked glycosylation
contributes to the discrepancy of the apparent molecular mass between
these two forms (13). Previous studies have indicated that
promoter-defined Elf-1 sites from different genes bind preferentially
different forms of Elf-1. The human IL-2 promoter-defined Elf-1 site
binds all forms of Elf-1 (14), whereas the enhancer of the
Moloney murine leukemia virus binds the 98-kDa form of Elf-1
(15).
We have reported previously that patients with SLE display aberrant
expression of several transcription factors including NF-
B
(16) and cAMP response element modulator
(17). These findings have indicated that transcription
factor expression and function is susceptible to adverse regulation in
T cells from SLE patients. Abnormal expression of transcription factors
should affect the transcription of respective target genes and
contribute to the pathogenesis of SLE. Because Elf-1 plays a critical
role in the transcription of the TCR -chain and other immune cell
genes (9, 14, 15), we conducted studies to determine the
expression of Elf-1 in patients with SLE. We demonstrate that nuclear
proteins from SLE T cells fail to bind to the TCR promoter-defined
Elf-1 sites either because the 98-kDa form of Elf-1 is not formed, or
because the formed 98-kDa Elf-1 lacks DNA-binding features.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty-one patients fulfilling at least 4 of the 11 revised criteria of the American College of Rheumatology for the classification of SLE (18) were studied. Twenty-seven of the patients were females and four were males with age range 21–80 years old (mean ± SD, 34 ± 11). Disease activity was measured using the SLE disease activity index (SLEDAI) score (mean ± SD, 7 ± 6.5). Patients were on various medications which included prednisone, hydroxychloroquine, azathioprine, and cyclophosphamide. Medications were discontinued 24 h before venipuncture. Fifteen normal volunteers served as normal controls.
Plasmids and reagents
The luciferase reporter constructs driven by either wild-type
(-307/+58) or mutant (FL
zEBS1+2, -307/+58 with both Elf-1 sites
mutated) TCR promoter have been described (9). Abs
against Elf-1, NF-B p50, TCR -chain, and actin were purchased
from Santa Cruz Biotechnology, (Santa Cruz, CA).
Purification of primary T cells and transfection
Primary T cells were obtained either by using magnetic beads to
remove all non-T cells (negative selection) as described before
(17, 19), or by incubating peripheral blood with
rosette/Ab complexes at room temperature for 20 min (StemCell
Technologies, Vancouver, British Columbia, Canada). The T cells
were then separated from the non-T cells by centrifugation at 2,400 rpm
for 25 min. The harvested T cells were washed and contaminated RBCs
lysed by ACK buffer (155 mM NH4Cl, 110 mM
KHCO3, 0.1 mM EDTA) if necessary. T cells were
transfected with the indicated plasmids using previously developed
protocols (17, 19). Briefly, freshly purified T cells were
incubated in medium containing 10% FCS and PHA (1 µg/ml) overnight.
Cells were then mixed thoroughly with the plasmids indicated and
subjected to electroporation at 0.25 V and 950 µF by using the
electroporator (Bio-Rad, Hercules, CA). Cells were harvested 
16 h
later and assayed for both luciferase and 
-galactosidase
activities.
EMSA
EMSA was performed as described previously (18).
The sequence of the oligonucleotides used for the DNA binding reaction
defined by the -147/-119 region of the TCR promoter was
5'-TCGAGAACCTCCAGGGCTTCCTGCCTGTGAACCA-3'.
Protein purification and Western blotting
The purification of cytoplasmic and nuclear proteins has been described before (17). Western blotting was performed following the manufacturer’s protocol (Amersham Pharmacia Biotech, Piscataway, NJ).
Preparation of mRNA, cDNA, PCR, and real-time PCR
One million T cells were used to extract RNA (RNA preparation
kit; Qiagen, Hilden, Germany). RNA was quantified and 500 ng of
total RNA was used for the preparation of cDNA by reverse transcription
(Reverse Transcription kit; Promega, Madison, WI). A total of 50 ng of
cDNA was used for each PCR. Primers were synthesized by Sigma-Aldrich
(St. Louis, MO). PCR beads were used for amplification (Amersham
Pharmacia Biotech). PCR products were separated on a 1.5% agarose gel
and the OD was analyzed using QuantityOne software (Bio-Rad) after
background subtraction from each band. The sequence of primers used for
PCR amplification are: 5'-GCGAGGGGGCAGGGCCTGCATGTGAAG-3',
5'-AGCCTCTGCCTCCCAGCCTCTTTCTGAG-3' (TCR -chain);
5'-CGGGGATGAAACAATTGAAACTAT-3', 5'-CTCGCTGGGTCCACTNTGATGTA-3'
(Elf-1); and 5'-CATGGGTCAGAAGGATTCCT-3', 5'- AGCTGGTAGCTCTTCTCCA-3'
(actin).
Chromatin immunoprecipitation analysis (ChIP)
T cells were treated by formalin fixation (1% final
concentration) for 10 min, followed by extensive washing. Cells were
then lysed and sonicated. The DNA-protein complexes were
immunoprecipitated using the indicated Ab and extracted by agarose
Sepharose beads (Santa Cruz Biotechnology). After several washing
steps, the cross-link between DNA and protein was reversed by
incubating the samples at 65°C for 4 h and the protein was
digested by protease K and the DNA was extracted (Qiagen DNA extraction
kit; Qiagen). The DNA was amplified with primers flanking the TCR
-chain promoter. DNA from 1 million cells was used per each PCR.
PCR products were run on a 2% agarose gel and quantified with
QuantityOne software. The sequence of the primers used for amplifying
the promoters are 5'-ACCTCGAGCCATCGAGAACT-3',
5'-ACAAGCTTCTTTCCCTCAGA-3' (TCR chain); and 5'-CTA AGT GTG GGC
TAA TGT AAC-3', 5'-TGT AAA ACT GTG GGG GT-3' (IL-2).
Isoelectric focusing gel (IEF) gel electrophoresis and Western blotting
Nuclear proteins were resolved in the IEF (Invitrogen, Carlsbad, CA) gel at 100 V for 1 h, 200 V for 1 h, and finally at 500 V for 30 min. The electrophoresed proteins on the gel were first equilibrated in the 0.7% acetic acid buffer at room temperature for 10 min and transferred to polyvinylidene fluoride membranes in the same buffer by applying 10 V for 1 h. The membrane was then air dried before blotting with Ab against Elf-1 as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter are necessary for the TCR
-chain gene expression in normal and SLE T cells
It was previously shown that Elf-1 is the transcription factor
which binds to two Elf-1 sites on the TCR -chain promoter in Jurkat
T cells. Mutagenesis of both Elf-1 sites down-regulated the
transcriptional activity of the promoter, whereas overexpression of
Elf-1 increased the activity of the promoter by three to four times in
COS-7 cells (9). To determine whether Elf-1 is similarly
required for the transcription of TCR -chain in primary human T
cells, we transfected luciferase reporter constructs driven by either
wild-type TCR -chain promoter (-307/+58), or by a promoter in which
both Elf-1 sites had been mutated (FLzEBS1+2) into normal and SLE T
cells. A construct which expresses -galactosidase was always
cotransfected into cells and served as the transfection efficiency
control. We first transiently transfected these constructs into Jurkat
cells and found that the wild-type promoter drives the expression of
luciferase four to five times higher than the mutated promoter, an
observation which is consistent with those reported previously
(9). We next transfected these constructs into T cells
from either normal (n = 2) or SLE patients
(n = 3). As shown in Fig. 1
, the promoter activity of the wild-type
TCR -chain promoter was four times higher in normal T cells than in
SLE T cells, indicating that the transcription of the TCR -chain
gene is defective in the latter. The reporter activity of the
constructs driven by the mutated FLzEBS1+2 promoter was lower than
the activity of constructs driven by the intact promoter, indicating
that the Elf-1 sites are important for the transcription of the TCR
-chain gene in both normal and SLE T cells. Decreased promoter
activity observed in these experiments was not due to differences in
the transfection efficiency between the normal and SLE T cells because
the expression levels of -galactosidase were comparable between
these two types of cells. Thus, the Elf-1-mediated transcriptional
defect is at least in part responsible for the defective expression of
the TCR -chain mRNA in patients with SLE that we reported previously
(2).
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Because we have failed to demonstrate any mutations in the TCR
-chain promoter in patients with SLE (20), we
considered that the decreased transcriptional activity in SLE T cells
should be due to abnormal expression and/or activation of Elf-1.
Accordingly, we investigated the expression pattern of Elf-1 in
patients of SLE. We purified nuclear proteins from normal and SLE T
cells and analyzed them by SDS gels electrophoresis and Western
blotting. As shown in Fig. 2a,
comparing to the normal controls, SLE patients (8 of 31) display
defective expression of the 98-kDa form of Elf-1 protein. In contrast,
the levels of the NF-B p50 (16) and the 80-kDa Elf-1
(Fig. 2b), which is enriched in the cytoplasm
(13), are similar among SLE patients and normal controls
(Fig. 2b). To establish whether global protein degradation
was responsible for the decreased expression of Elf-1 in SLE T cells,
we subjected nuclear proteins to SDS gel electrophoresis and staining
with Coomassie blue (Fig. 2c). We found that while each
sample varies slightly in the intensity of individual bands, there was
no extensive protein loss in SLE patients. To this end, we compared the
total nuclear protein expression levels between normal and SLE T cells
by densitometry and calculated the average intensity of five different
bands representing proteins of various molecular mass (Fig. 2c, 2 days). We observed that proteins from SLE T cells are
decreased by 30–40% in bands a and e, while they have comparable
levels in bands b–d, and NF-B p50. It should be noted that the
intensity of band c, which represents proteins with molecular mass
close to the 98-kDa Elf-1, is comparable between normal and lupus T
cells. These data indicate that global protein degradation does not
play a significant role in the decreased expression of the 98-kDa Elf-1
in SLE patients, and that the loss of 98-kDa Elf-1 involves additional
mechanisms.
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b), we performed RT-PCR to rule out additional defects in
the transcriptional expression of the Elf-1 gene. As shown
in Fig. 3, the levels of Elf-1 mRNA are
comparable in SLE (n = 18) and normal T cells
(n = 10).
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-chain in patients of SLE
To determine whether the decreased expression of the 98-kDa form
of Elf-1 is associated with decreased DNA binding, we incubated nuclear
proteins from normal (n = 15) and SLE
(n = 31) T cells with an oligonucleotide which encodes
the Elf-1-binding sites on the TCR -chain promoter. As shown in Fig. 4b, low levels of nuclear
98-kDa Elf-1 in SLE patients were associated with decreased intensity
of the band that reflected the binding of Elf-1 to DNA. These
experiments confirmed that the loss of the 98-kDa Elf-1 is specifically
associated with the loss of binding to the TCR -chain promoter. We
then performed Western blots to determine whether low DNA-binding
activity is associated with decreased TCR -chain expression. As
shown in Fig. 4c, loss or low binding of SLE T cell nuclear
proteins to the Elf-1 site of the TCR -chain promoter is associated
with decreased expression of TCR -chain.
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-chain
In the course of these experiments, we observed that a group of
SLE patients (14 of 31) displayed low DNA-binding activity despite the
fact that they expressed normal levels of the 98-kDa form of Elf-1
(Fig. 5a, comparing lupus
patients 2, 3, and 4 to the normal controls). As shown in Fig. 5a, low DNA-binding activity resulted in low expression of
TCR -chain. This observation indicated that although the DNA-binding
form of Elf-1 resided in the 98-kDa form, in certain patients the final
active form was not produced.
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b, decreased DNA
binding was noted in protein extracts from SLE T cells, which express
high levels of both 80- and 98-kDa Elf-1, suggesting that the 80-kDa
Elf-1 does not bind the TCR -chain promoter. The expression levels
of TCR -chain in T cells from this group of patients reflected
accurately the intensity of the band reflecting protein binding to the
Elf-1 oligonucleotide (Fig. 5b).
Elf-1 does not bind to the TCR -chain promoter in live T cells
from patients with SLE
Shift assays determine in vitro protein-DNA interactions and may
not reflect in vivo binding. To ascertain that Elf-1 fails to bind to
the TCR -chain promoter in live SLE T cells, we subjected T cells to
formaldehyde fixing, sonication, and immunoprecipitation of TCR
-chain promoter with anti-Elf-1 Ab followed by RT-PCR which
specifically amplifies the TCR -chain promoter. We elected to
perform these experiments using cells from SLE patients
(n = 4) who were shown to have comparable levels of
98-kDa Elf-1 along with T cells from normal donors (n =
3) (Fig. 6a). As shown in Fig. 6b, the SLE samples displayed a significant decrease in the
in vivo binding of Elf-1 to the TCR -chain promoter (four of four).
Because it is known that Elf-1 binds to the IL-2 promoter
(14), we performed ChIP experiments using anti-Elf-1
Ab and PCR primers spanning the -232/-55 region of the IL-2 promoter.
As it can be seen in Fig. 6b, Elf-1 binds to the IL-2
promoter at comparable levels between normal and SLE T cells. This
experiment serves also as an additional control for the
immunoprecipitation and PCR amplification steps of the ChIP assays used
above to establish that Elf-1 from SLE T cells fails specifically to
bind to the TCR -chain promoter. Furthermore, the same SLE samples
displayed decreases in the binding to Elf-1 oligonucleotides in in
vitro shift assays (Fig. 6c), confirming the results from
the in vivo ChIP assays. The consequence of this defective TCR
-chain promoter binding by Elf-1 in this group of SLE patients is
reflected by the decreased levels of TCR -chain mRNA, as assayed by
RT-PCR on the total RNA collected from the same patients (Fig. 6d). The levels of both Elf-1 and actin mRNA were comparable
between normal controls and SLE patients. Altogether, our in vitro and
in vivo data indicate that defective DNA binding of Elf-1 to the TCR
-chain promoter in SLE T cells is associated with defective
transcription of TCR -chain.
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Previously, we had noted (13) that the nuclear
abundant 98-kDa Elf-1 is further separated into three distinct
subcomponents in 6% SDS gels. The low binding of SLE T cell nuclear
extracts to the TCR -chain promoter, despite normal levels of 98-kDa
Elf-1 form, suggested that it is different from the normal 98-kDa
Elf-1. To determine the nature of the difference between normal and SLE
T cell-expressed 98-kDa Elf-1, we subjected normal T cell nuclear
proteins to IEF gel analysis along with nuclear extracts from T cells
from SLE patients. The used SLE samples expressed slightly higher
levels of 98-kDa Elf-1 but less DNA-binding activity (Fig. 7, a and b). IEF
can detect protein modifications which do not contribute significant
changes in the molecular size of the protein, and thus cannot be
detected by SDS gel electrophoresis. As shown in Fig. 7c,
normal T cell nuclear proteins are composed of three subcomponents
(five of five), a finding consistent with our previous observation
using 6% SDS gel electrophoresis (13). These three bands
are widely separated in the IEF gel, indicating that the individual
components are the products of distinct posttranslational
modifications. Interestingly, lupus patients lacked the subcomponent
migrating between the two other major subcomponents (four patients of
seven studied). Whether the missing band is the major DNA-binding
component needs to be determined.
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-chain
promoter correlates with the levels of TCR -chain mRNA
To obtain an overall picture of the relationship between nuclear
protein DNA binding and the expression of TCR -chain, we plotted the
densitometry readings obtained from either shift assays or ChIP against
the -actin-normalized TCR -chain mRNA levels obtained from
RT-PCR. As it can be seen in Fig. 8, there is a strong correlation (r = 0.51,
p = 0.05) between DNA protein binding and -chain
expression, further proving that the defective expression of TCR
-chain in SLE T cells is the result of decreased TCR -chain gene
transcriptional activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain promoter is
decreased in patients with SLE because the levels of TCR -chain mRNA
in SLE T cells are decreased (2), and because the activity
of a reporter construct driven by the TCR promoter transiently
transfected into SLE T (Fig. 1) cells is decreased. In this study, we
have demonstrated that the two Elf-1 sites defined by the TCR -chain
promoter are critical for the transcription of TCR -chain not only
in Jurkat cells (9), but also in primary T cells from both
normal individuals and SLE patients. The fact that the TCR -chain
promoter of SLE patients does not express any mutations or
polymorphisms (20), which could explain defective
transcriptional activity, prompted us to search for defects in the
expression and/or activation of the Elf-1 transcription factor. After
establishing that the Elf-1 message levels in SLE T cells is comparable
to those in normal T cells (Fig. 3) and that the main 80-kDa
cytoplasmic form of Elf-1 (13) was present at normal
levels in SLE T cells (Fig. 2b), we searched for defects in
the posttranslational processes that are responsible for the generation
of the nuclear 98-kDa form, which binds to the TCR promoter and
controls its transcriptional activity.
Elf-1 cDNA encodes a peptide of 619 aa and should migrate in SDS gel as
a protein of 68 kDa. Interestingly, Elf-1 in the cytoplasmic protein
fraction migrates mainly as an 80-kDa protein, while the dominant form
in the nucleus migrates as 98 kDa. We have reported that the 98-kDa
form is the form of Elf-1 that binds to the TCR -chain promoter
(13). This conclusion was based on the fact that the
intensity of the nuclear protein binding to the TCR -chain
oligonucleotides correlated with the intensity of the 98-kDa form, but
not with that of the 80-kDa form as determined by Western blot analysis
(Figs. 4 and 5b). In addition, UV cross-linking experiments
using a labeled Elf-1 defined oligonucleotide and nuclear proteins from
normal T cell produced a band expected from the binding of the 98-
rather than the 80-kDa protein (data not shown).
Two patterns of Elf-1 activation defects were identified in SLE T cells
in this study. In the first, the 98-kDa form was absent probably
because of defective phosphorylation and O-glycosylation,
two processes shown to be responsible for the discrepancy between the
predicted and the observed molecular mass of Elf-1 (13).
In the second, although the apparent 98-kDa form is generated at normal
levels as detected by Western blot analysis (
Figs. 5–7), it failed to
bind to the TCR promoter-defined Elf-1 sites. Decreased DNA binding
was established in EMSA using TCR -chain-defined Elf-1
oligonucleotides (
Figs. 5–7) and ChIP (Fig. 6), which enables
detection of transcription factors in live cells. Because the normal
nuclear Elf-1 could be resolved in to three components in IEF gels, and
the SLE nuclear 98-kDa form missed one of the three components, we
suspect that the missing band is responsible for the defective DNA
binding.
Transcription factor abnormalities seem to be extensive and
diverse in patients with SLE. In an earlier study, we had demonstrated
that the NF-B activity was decreased in SLE T cells because of
decreased or absent levels of the p65 subunit (16).
Interestingly, the p50 subunit was not affected, suggesting that the
decrease of p65 chain (16) and of the Elf-1 98 chain
reported herein do not represent generalized T cell protein
degradation. In support of this conclusion is the finding that the
transcriptional repressor cAMP response element modulator is
increased in T cells from patients with SLE (17). The data
shown in Fig. 2 also support the conclusion that T cell proteins do not
undergo indiscriminate degradation. However, it should be noted that
under certain conditions T cell proteins may be degraded as is the case
of caspase-mediated degradation of the TCR -chain in Jurkat T cells
(8).
We have shown that the discrepancy between the predicted molecular mass
(68 kDa) and the observed mass of the 98-kDa form of Elf-1 in normal T
cells is at least in part the result of phosphorylation and
O-linked glycosylation (13).
O-linked glycosylation has been shown to contribute to the
activation of a number of transcription factors (21).
Defective glycosylation may also be responsible for the decreased
conversion of the 68-kDa because phosphorylation alone cannot account
for the 38-kDa difference between the predicted and the observed
molecular mass of Elf-1. At least 20 Elf-1 amino acid residues (11 Thr
and 9 Ser) can be identified on Elf-1, which have high possibility
(0.75–0.98, which is above the threshold) for O-GlcNAc
modification. Deficiency in
1,6-N-acetylglucosaminyltransferase V, an enzyme in the
N-glycosylation pathway, has been shown to cause kidney autoimmune
disease and increased susceptibility to autoimmune encephalitis
(22), indicating that glycosylation defects may be
associated with autoimmunity. It is unclear at this moment whether
defective glycosylation or phosphorylation, or both, are responsible
for the decreased expression of the 98-kDa form. We have accumulated
evidence that the cellular levels of the 98-kDa Elf-1 represent the
balance between the phosphorylation and dephosphorylation of
Elf-1 (Ref. 13 and our unpublished observations). Protein
kinase C (PKC) activity has been claimed to be decreased in
patients with SLE (23). Defective PKC activity may be
responsible for the decreased formation of the 98-kDa Elf-1 since we
showed before that forced expression of active form of PKC 
and 
enhanced the expression of the 98-kDa Elf-1 in Jurkat cells.
Alternatively, increased phosphatase activity in SLE T cells can
account for the decreased expression of the 98-kDa form. Indeed, we
have found that treatment of T cell protein extracts with bacterial
phage 
phosphatase (13) results in the disappearance of
the 98-kDa form. A dephosphoryalted 98-kDa Elf-1 may undergo
degradation (13). It should be noted that phosphorylation
of c-Jun has been shown to stabilize the protein and protect it from
degradation through ubiquitin pathway (24).
The observation that in more than a third of the studied SLE patients
(14 of 31) there was a significant decrease in the TCR -chain
promoter binding despite the fact that the apparent levels of the
98-kDa protein were normal presented a special challenge. In previous
studies (13), we had observed that the 98-kDa Elf-1 could
be resolved in 6% SDS gels into three components, which became more
obvious when the cells were treated with the phosphatase inhibitor
okadaic acid. These studies prompted us to consider that in certain SLE
patients, not all components are formed as a result of defective
phosphorylation. Indeed, separation of normal nuclear 98-kDa Elf1 in
IEF gels produced three distinct bands, and SLE samples were shown to
miss one of them. Additional studies are needed to confirm that the
middle band of the Elf-1, which is missing in SLE T cells, represents
the one responsible for the DNA binding.
It is possible that other nuclear proteins, particularly members of the
Ets family which share the Ets DNA binding domain with Elf-1 and thus
recognize similar DNA binding sites, replace Elf-1 and play a role in
the regulation of the TCR -chain expression in SLE T cells. However,
our EMSA revealed that in SLE patients, the nuclear protein/DNA
interaction generated only one band which comigrated with the
Elf-1-specific DNA protein complex in a way similar to that observed in
the normal T cells (
Figs. 4–6). The lack of additional bands in EMSA
using SLE T cells suggests the absence of binding of other Ets family
proteins. It should be noted that the T cell-abundant Ets-1 and 2 have
molecular mass of 50 and 56 kDa, respectively; and therefore, if
present, they should generate shifted bands with different mobility
features than the 98-kDa Elf-1. Furthermore, unlike transcription
factors such as CREB/cAMP response element modulator, which
frequently either coexist or compete for the same binding sites,
members in the Ets family display unique binding preference in a
variety of gene promoters (14).
We failed to establish a correlation between the defective expression
and DNA binding of Elf-1 with disease activity or treatment. We
considered the possibility that the observed defects are due to the
medications that the patients were receiving during the performance of
this study and in particular of prednisone since most of the SLE
patients were receiving 10 mg of prednisone per day. We have calculated
the blood level of prednisone and found that shortly after it is taken
(2–3 h), it reaches its peak blood level which is approximately
equivalent to 70 nM of dexamethasone. It stays at its peak level only
for short period of time and then it declines rapidly to 7 nM. We
have previously determined that culture of normal T cells with 10 nM of
dexamethasone increases rather than decreases the expression of
-chain (25). Therefore, we believe that treatment with
prednisone cannot account for our observations. It should be noted that
higher concentrations of dexamethasone (100 nM) can suppress the
expression of -chain (26). Defective Elf-1 expression
was observed in patients with a wide variation in SLEDAI scores.
Reproducibly decreased levels of Elf-1 protein and DNA-binding levels
were observed in patients (n = 3) studied several times
over a period of 2 years in whom the SLEDAI scores fluctuated.
Specifically, in one patient the 98-kDa Elf-1 was undetectable on four
different occasions, and in two more patients, who expressed normal
levels of 98-kDa Elf-1, the DNA-binding intensity remained similarly
low on two different occasions (data not shown).
It is important to note that although this study has presented a strong
correlation between the Elf-1/DNA interaction and the mRNA levels in
SLE T cells, the expression levels of the TCR -chain protein in a
portion of lupus patients (5 of 31, 16%) did not reflect accurately
the TCR -chain promoter activity (data not shown). Because previous
studies have shown that TCR -chain internalization and subsequent
degradation also plays a role in the down-regulation of TCR -chain,
further studies are needed to explore additional mechanisms that may
contribute to the decreased expression of -chain in SLE T cells.
In conclusion, we have established that defective transcriptional
activity of the TCR -chain promoter contributes to the decreased
expression of TCR -chain mRNA and protein. More importantly, we have
demonstrated that defective activation, rather than transcription and
translation of Elf-1, the sole transactivating factor known to be
involved in the transcription of the TCR -chain gene, is responsible
for decreased TCR -chain expression. The exact ranking of our
findings in terms of their importance in the pathogenesis of human SLE
is unknown. However, it appears that aberrations at the level of
transcription factor must be more central to the origin of the disease.
It is also certain that the complete characterization of all molecular
defects displayed by SLE T cells may permit the identification of a
"molecular signature" for each patient or group of patients
with SLE.
The repercussions of our findings extend beyond the expression of TCR
-chain because Elf-1 has been implicated in the transcriptional
regulation of additional genes that are relevant to the regulation of
the immune response such as blk, lck, and
lyn kinases (27), IL-2R -chain
(28), CD4 (29), GM-CSF (12), and
IL-2 (14). Although the form of Elf-1 that binds to the
promoters of these genes may not be the same as the one that binds to
the TCR -chain promoter, we suspect that the identified defective
Elf-1 activation in SLE T cells may account for additional defects in
these cells (1).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yuang-Taung Juang, Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910. E-mail address: Yuang-Taung.Juang{at}NA.AMEDD.ARMY.MIL
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; ChIP, chromatin immunoprecipitation analysis; IEF, isoelectric focusing gel; PKC, protein kinase C.
Received for publication July 25, 2002. Accepted for publication September 16, 2002.
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