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INTRODUCTION |
Fas ligand (FasL)1 is a
Type II trans-membrane protein that belongs to the tumor necrosis
factor family of cytokines and induces apoptosis in cells expressing
the Fas receptor (Fas, CD95, APO-1) (for reviews, see Refs. 1-4). The
Fas/FasL system was first identified in T cells (5, 6) where it plays a
key role in eliminating T cell populations following antigenic
stimulation and clonal proliferation. T cell activation leads to
expression of both Fas and FasL, allowing contact-induced death via
apoptosis (5, 7). The regulation of FasL gene expression in T cells is
currently being elucidated (8-12). More recently, it has become
apparent that the Fas/FasL system is functional in a variety of other
cell types including many in which expression of these proteins is constitutive rather than inducible (summarized in Ref. 4). Of
particular interest is the observation that FasL is constitutively expressed by cells in "immune privileged" sites such as testicle (13), eye (14), placenta (15, 16), and brain (17) (also see Ref. 18).
In addition, several malignant cell lines and primary malignancies have
now been shown to express FasL (4, 17, 19-26), and it is postulated
that this is important for invasive growth, as the tumors are rendered
immune privileged.
Recent cloning of the FasL enhancer-promoter region has enabled
investigation of the transcriptional regulation of FasL, with initial
studies focusing on the inducible regulation of FasL transcription following T cell activation (8, 10, 11). In T cells,
Ca2+-dependent signals, via calcineurin, lead
to an increase in the nuclear concentration of the transcription factor
(NFAT) which binds to the promoter/enhancer region of the FasL gene and
increases its transcription (8, 11). However, it remains unknown what regulates basal non-inducible FasL gene transcription, specifically in
cells known to constitutively express high levels of FasL and maintain
an immune-privileged environment.
In this study, we have investigated the transcriptional regulation of
FasL in a Sertoli cell line (TM4) that is known to have constitutive
FasL expression (10), and compared it to Jurkat T cells in which FasL
gene transcription can be induced by specific stimuli. Results from
this study demonstrate that a segment of the 5'-untranslated region of
the FasL gene located between
318 and
237 relative to the
translation initiation site is important for constitutive basal FasL
gene transcription, and that an Sp1 but not an NFAT or NFKB consensus
DNA-binding site within this region is necessary and sufficient for
transcription in both Sertoli and Jurkat T cells. In addition, nuclear
extracts of Sertoli cells contain high levels of Sp1 and Sp3 that
specifically bind to the GGGCGG consensus sequence present in the FasL
gene. Overexpression of Sp1 but not Sp3 leads to increased
transcription from the FasL promoter region through this specific motif
in both Sertoli and Jurkat T cells. This data demonstrates that the
basal constitutive FasL transcription is mainly dependent on Sp1 in
these different cell types demonstrating that the control of inducible
and constitutive FasL gene transcription are mediated by separate
transcription factors.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Reagents--
The testicular murine Sertoli cell
line (TM4) was purchased from American Type Culture Collection (ATTC)
and cultured in Dulbecco's modified Eagle's medium/F-12 medium
(Biowittaker) supplemented with 5% horse serum (Biowittaker), 2.5%
fetal bovine serum (Intergen), 1% glutamine, 1% streptomycin, and 1%
penicillin. The Jurkat T cell line was purchased from ATCC and cultured
in RPMI medium (Life Technologies, Inc.) supplemented with 5% fetal
bovine serum (Intergen), glutamine, and antibiotics as above.
Anti-Sp1(PEP2)X, anti-Sp3(D-20)-GX, anti-NFATc(K-18)X, and
anti-STAT3(c-20)X antibodies were purchased from Santa Cruz
Biotechnology Inc. Anti-HA antibodies were purchased from Boehringer
Mannheim and unconjugated anti-rat IgG(H+L) antibodies were purchased
from Pierce.
Plasmids and Oligonucleotides--
Plasmids containing FasL
promoter deletion mutants were cloned upstream of the firefly
luciferase reporter gene (FasL-Luc-0 through FasL-Luc-6) and have been
previously described (8). The luciferase reporter gene, pGL2-basic
(pGL2-B), was purchased from Promega. Point mutations targeted three
separate transcription factor consensus sequences within the FasL
promoter region of FasL-Luc-3 and were created using polymerase chain
reaction-based techniques as described previously (8). The resultant
products were cloned into pGL2-B, and the final constructs sequenced to verify the appropriate nucleotide mutations. The mutations introduced are shown in Fig. 1. The FasL-Luc
constructs are shown in Fig. 2. The
NFKB-dependent luciferase reporter gene (NFKB-Luc) and the
IKB
(S32/36) have been previously described (27, 28). The thymidine
kinase-(TK)-
-galactosidase (
-gal) reporter gene (TK-
-gal) was
purchased from CLONTECH. pcDNA3 was purchased
from Invitrogen. The Sp1 and Sp3 expression vectors have been described (29) and were provided by Dr. Horowitz, North Carolina State University, Raleigh, NC.

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Fig. 1.
Fas ligand promoter sequence showing the
mutated forms used in transfections and EMSAs. A segment of the
wild-type FasL promoter sequence is shown in the top line,
with three transcription factor consensus sequences (Sp1, NFAT, NFKB)
and their mutated forms ( Sp1, NFAT, NFKB, NFAT- NFKB,
Sp1- NFAT- NFKB) shown below. The point mutations are
underlined in each sequence. The Sp1, NFAT, and
NFKB mutations are present in the oligonucleotides used in EMSA
experiments (each oligonucleotide spans bases 288 to 263) and the
FasL-Luc-3 deletion constructs used in transfections.
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Fig. 2.
Fas ligand promoter deletion constructs used
in transfections. 5' deletions of the FasL promoter/enhancer DNA
sequence were cloned upstream of the luciferase (Luc) reporter gene in
pGL2-B. Numbers indicate nucleotide locations with respect
to the translational start site of the FasL gene. An arrow
indicates the transcription initiation site. A black box
indicates the location of DNA binding motifs for three transcription
factors: Sp1, NFAT, NFKB. Plasmid designations are given at the
right.
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Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared from Sertoli cells as described previously (27).
Briefly, the cells were scraped off of the flask in cold
phosphate-buffered saline, washed in buffer A (10 mM Hepes,
1.5 mM MgCl2, 10 mM KCl), and then
lysed two times in buffer A containing protease inhibitors (aprotinin
at 0.4 µg/ml, leupeptin at 0.002 mg/ml, pepstatin at 0.8 µg/ml, and
phenylmethylsulfonyl fluoride at 0.175 µg/ml), 100 µM
dithiothreitol, and 0.1% Nonidet P-40. Nuclei were pelleted, washed in
Buffer A, and the nuclear protein extracted in Buffer C (20 mM Hepes, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 100 µM dithiothreitol, and protease inhibitors as above).
After pelleting the nuclear debris, the supernatant was removed and
diluted with twice the volume of Buffer D (20 mM Hepes,
20% glycerol, 50 mM KCl, 0.2 mM EDTA, 100 µM dithiothreitol, and protease inhibitors as above).
Protein concentration was calculated using the Bradford method
(Bio-Rad). Oligonucleotides were annealed to make double-stranded
target DNA by successive incubations: 88 oC for 2 min,
65 oC for 10 min, 37 oC for 10 min, room
temperature for 10 min. The DNA was then end labeled with
[
-32P]ATP using polynucleotide kinase
(37 oC for 30 min, 65o for 10 min) and the
unincorporated radioactive nucleotides removed on a column of
Spectra/GelTM ACA 202 (fractionation range 1,000-15,000)
beads (Spectrum). Binding reactions were done by incubating the labeled
DNA with nuclear extract in binding buffer (10 mM Tris-HCl,
pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM EDTA, pH
7.5, 0.1% Nonidet P-40) at room temperature for 15 min. Reactions
using antibodies were performed as above, with a final addition of
antibody and incubation on ice for 30 min. The reactions were separated
by polyacrylamide gel electrophoresis (5%), the gel dried and exposed
to autoradiographic film.
Western Blots--
Twenty µg of protein from whole Sertoli
cell lysates were loaded per well, separated in SDS-polyacrylamide gel
electrophoresis (10%), and transferred to Immobilon-P membrane by
electrophoresis. The membrane was then blocked with 5% milk overnight,
incubated with anti-HA antibodies (100 ng/ml for 1 h), anti-rat
IgG (H+L) (1:10,000 for 30 min), and horseradish peroxidase (1:10,000
for 30 min), and developed using the ECL system (Amersham) for 2 min. Three washes were done between each step, all in Tris-buffered saline
with Tween 20 for 10 min.
Transfections--
Gene transfections in Sertoli cells were done
in duplicate using LipofectAMINE (Life Technologies, Inc.) as per the
manufacturer's instructions. Sertoli cells were plated in 6-well
tissue culture dishes at 0.9 × 105 cells per well,
grown for 48 h prior to the addition of DNA suspension, and
incubated for 24 h following gene transfection. In all cases, the
total amount of DNA in each transfection was kept equal by the addition
of control DNA (pcDNA3) where appropriate. The luciferase (Luc) and
-galactosidase (
-gal) activity were read using the Dual-LightTM chemiluminescence system following the
manufacturer's instructions (Tropix, Inc.). Briefly, the cells were
washed twice in phosphate-buffered saline and total cell extracts
prepared in 250 µl of lysis buffer. The luciferase activity of 5 µl
of extract was read using a luminometer (Lumat LB9501). The solutions
were left at room temperature for 1 h before the
-gal activity
was read using the same luminometer. All experiments were repeated at
least twice. Reporter gene activity is reported as relative light units
which results from the ratio of luciferase units to
-gal units for
each data point. Error bars on transfection data represent the standard
deviations of duplicate samples. Controls performed in all transfection
experiments included transfection of control DNA (pcDNA3) alone,
and the empty luciferase reporter vector (pGL2-B) with the TK-
-gal
reporter plasmid. In each control case, the luciferase activity was
detectable, minimal, and significantly below all other data points.
Gene transfections in Jurkat T cells were done using
FuGeneTM6 Transfection Reagent (Boehringer Mannheim) as per
the manufacturer's instructions. DNA-FuGene mixtures were incubated
for 48 h with cells washed in phosphate-buffered saline and split
equally for luciferase and chloramphenicol acetyltransferase (CAT)
assays. Luciferase activity was determined using the "Luciferase
Assay System" (Promega) following the manufacturer's instructions.
CAT activity was determined using an enzyme-linked immunosorbent assay (Boehringer Mannheim) following the manufacturer's instructions. For
Jurkat T cells, relative light units were calculated by normalizing the
luciferase with the CAT units.
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RESULTS |
Constitutive Activity of the FasL Enhancer-promoter Region in
Sertoli Cells Is Mediated Through an 80-Base Pair 3' Region--
To
determine which segment(s) of the 5'-untranslated region of the FasL
gene is important for transcription in Sertoli cells, these were
transfected with the FasL gene promoter constructs outlined in Fig. 2,
and activity from the luciferase reporter gene measured. The results
shown in Fig. 3 indicate that
transcription from the FasL-Luc-0 construct was detectable, but strong
and increasing transcriptional activity was detected from FasL-Luc 1 to
3, with maximal activity provided by the FasL-Luc-3 reporter gene. The transcription activity then dropped sharply, and continued to decrease
from FasL-Luc-4 through FasL-Luc-6. These results indicate that an
approximate 80-base pair segment of the 5'-untranslated region of the
FasL gene located between
318 and
237 relative to the translation
initiation site is involved in promoting transcription in TM4 Sertoli
cells. This region, specifically that between
280 to
267 base
pairs, contains three DNA motifs known to bind the transcription
factors Sp1 (GGGCGG), NFAT (GGAAA), and NFKB (GAAACTTCC) (see
Fig. 1). It should be noted that the NFKB sequence is an imperfect
consensus sequence, and will be referred to as NFKB-like. Although
construct FasL-Luc-0 contains the stretch of three transcription factor-binding sites, the transcriptional activity of this reporter gene was barely above background, possibly suggesting the presence of a
DNA sequence upstream from
463 to which transcription factors with
repressor activity bind.

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Fig. 3.
Baseline FasL promoter activity in Sertoli
cells. Sertoli cells were transfected with the FasL-Luc reporter
gene constructs (FasL-Luc-0 through FasL-Luc-6) as shown in Fig.
2.
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An Sp1 DNA Binding Motif Is Required for Effective Constitutive
Transcription from the FasL Promoter in Sertoli Cells--
Using the
FasL-Luc-3 construct (Fig. 2), point mutations were introduced into
each of three transcription factor DNA binding motifs, as shown in Fig.
1. These constructs were then transfected into Sertoli cells, and
transcription from the FasL gene promoter measured by quantifying the
activity of the luciferase reporter genes. The results shown in Fig.
4 demonstrate that point mutations introduced into the Sp1 binding motif resulted in the most important reduction of basal transcription from the FasL gene. A less significant reduction was observed when the NFAT or NFKB sites were separately mutated (Fig. 4). Because of the small but detectable drop of the basal
FasL gene transcription when the NFKB-like motif was mutated, we
investigated whether the combination of mutation of the Sp1, NFAT, and
NFKB-like motifs would be additive. As shown in Fig. 4, the combined
mutation of these cis-acting motifs decreases only moderately the basal
FasL gene transcription beyond that observed with the single Sp1
mutation. This data suggests that the Sp1 cis-acting motif alone is
dominant in conferring the basal FasL transcription in Sertoli cells.
Nevertheless, and because of the reported interaction between Sp1 and
NFKB (30), we intended to further confirm or exclude the role of the
NFKB-like motif as one that may confer basal FasL gene transcription
independently from Sp1. For this, we tested the role of an IKB
expression vector (in which Ser32/36 have been mutated to
alanine) which has previously been shown to inhibit both basal and
inducible NFKB activity (28). Sertoli cells were transfected with the
FasL-Luc-3 wt or
NFKB construct together or not with the IKB
32/36A expression vector and the TK-
-gal expression vector to
normalize for transfection efficiency. As shown in Fig.
5A, the expression of IKB
32/36A does not interfere with the basal activity of the FasL-Luc-3 wt
or
NFKB. However, in the same transfection experiment in Sertoli
cells, IKB
32/36A interfered with both the basal and inducible
(tumor necrosis factor) transcriptional activity of a NFKB-Luc reporter
gene (Fig. 5B). Altogether, this data implies that the Sp1
cis-acting motif controls the basal activity of FasL in Sertoli
cells.

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Fig. 4.
Mutations of transcription factor DNA binding
motifs within the FasL promoter decrease transcription in Sertoli
cells. Sertoli cells were transfected with the wild-type
FasL-Luc-3 construct and FasL-Luc-3 constructs containing point
mutations in one or more of the three transcription factor DNA binding
motifs shown in Fig. 1: Sp1, NFAT, NFKB, NFAT- NFKB, and
Sp1- NFAT- NFKB.
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Fig. 5.
The NFKB-like site in the FasL promoter does
not contribute to FasL expression in Sertoli cells by binding NFKB.
A, Sertoli cells were co-transfected with either the
FasL-Luc-3 wild-type reporter construct or with mutations in the
NFKB-like DNA binding motif (FasL-Luc3- NFKB; see Fig. 1), and the
IKB 32/36A (IKB ) expression vector. B,
Sertoli cells were co-transfected with a luciferase reporter construct
containing three NF B sites (NFKB-Luc) and IKB 32/36A
(IKB ).
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Sp1 Present in Nuclear Extracts of Sertoli Cells Binds the Sp1 DNA
Binding Motif of the FasL Promoter--
After demonstrating that the
Sp1 DNA motif is required to convey basal transcription from the FasL
promoter, we wanted to identify the composition of nuclear proteins
that interact with this cis-acting motif. Using EMSA (Fig.
6A, lanes 1 and 5),
we observed that nuclear extracts from Sertoli cells contain three protein complexes that bind an oligonucleotide which contains the Sp1,
NFAT, and NFKB DNA binding motifs and encompasses
288 to
263 base
pairs relative to the translation initiation site (Fig. 1). These DNA
binding complexes were further analyzed using specific antibodies
directed to Sp1, Sp3, NFAT, NFKB, and STAT-3. The results shown in Fig.
5A demonstrate that the slower migrating protein complex (a)
contains Sp1, while the two faster migrating complexes (b and c)
contain Sp3. We did not demonstrate what constitutes the difference in
mobility seen between complexes b and c. None of the complexes
contained NFAT, STAT-3, or NFKB (NFKB; data not shown).

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Fig. 6.
Sp1 and Sp3 from Sertoli cells bind the Sp1
DNA binding motif of the FasL promoter. A, EMSA was
performed using nuclear extracts from Sertoli cells and a labeled
oligonucleotide containing bases 288 to 263 of the wild-type
promoter sequence of FasL (see Fig. 1). Antibodies to transcription
factors were added to some of the binding reactions as indicated. The
three un-shifted protein complexes observed to bind the wild-type
oligonucleotide (lanes 1 and 5) are labeled from
largest to smallest as a, b, and c. B,
EMSA was performed using nuclear extracts from Sertoli cells and
oligonucleotides spanning bases 288 to 263 of the FasL promoter.
The three protein complexes identified are labeled from largest to
smallest as a, b, and c. Lanes 1-4 show the
results of binding reactions using radioactively labeled (*)
oligonucleotides containing the wild-type and mutated sequences shown
in Fig. 1. Lanes 5-8 show the results of binding reactions
in which nuclear extracts were preincubated with un-labeled, mutated
oligonucleotides prior to the addition of labeled (*) wild-type
oligonucleotide.
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To confirm that Sp1 and related family members constitute the major
portion of nuclear protein binding to the oligonucleotide encompassing
bases
288 to
263 relative to the FasL translation start site,
Sertoli cell nuclear extracts were incubated with three separate
oligonucleotides, each containing point mutations in one of the three
transcription factor DNA binding motifs:
Sp1,
NFAT, and
NFKB
(see Fig. 1). When the oligonucleotides are used in EMSA (Fig.
6B), only mutations introduced into the Sp1 cis-acting motif
eliminated DNA binding of the protein complex containing Sp proteins
(Fig. 6B, panel 1). In addition, incubation with an excess
of unlabeled oligonucleotide containing mutations in either NFAT of
NFKB competed away all DNA binding activity of the protein complexes
bound to the 32P-labeled wild type
288 to
263 FasL
oligonucleotide. An excess of unlabeled oligonucleotide containing
mutations in the Sp1 site did not compete with the DNA binding of the
protein complex (Fig. 6B, panel 2). These results indicate
that the nuclear protein complex present in Sertoli cells, which binds
to the putative SP1 cis-acting sequence, contains at least Sp1 and Sp3.
Overexpression of Sp1 but Not Sp3 in Sertoli Cells Increases
Transcription from the FasL--
After showing that the Sp1 cis-acting
DNA motif is necessary for the constitutive transcription of the FasL
promoter in Sertoli cells, and that Sp1 and Sp3 present in nuclear
extracts are major components of the nuclear protein complex that binds
to this motif, we next investigated whether overexpression of Sp1 or
Sp3 would modify the transcriptional regulation of the FasL promoter.
Expression of Sp1 and Sp3 in Sertoli cells was first verified by
performing transient transfections with Sp1 and Sp3 expression vectors
in Sertoli cells followed by analysis of the transfected cell lysates in immunoblotting assays with anti-Sp1 or Sp3 antibodies. As shown in
Fig. 7A, significant levels of
both Sp1 and Sp3 can be detected. Fig. 7B shows the effect
of Sp1 and Sp3 overexpression on the transcriptional activity of the
FasL promoter in Sertoli cells. Cells were transfected with the
FasL-Luc-3 or FasL-Luc-3-
Sp1 constructs plus either the Sp1 or Sp3
expression vector, or both. The results demonstrate that overexpression
of Sp1 alone leads to increased transcription from the wild-type FasL
promoter, but not from the FasL promoter containing the mutated Sp1
cis-acting sequence. Overexpression of Sp3 alone has essentially no
transcriptional effects on the wild type or mutated FasL promoter, and
its co-expression with Sp1 does not modify the observed effects of Sp1
alone. These results demonstrate that Sp1 alone is sufficient to
enhance the basal transcription from the FasL promoter through the Sp1
cis-acting sequence in Sertoli cells.

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Fig. 7.
Sp1 and Sp3 expression vectors produces Sp1
and Sp3 protein in Sertoli cells. Sp1 increases transcription of
the FasL gene, Sp3 does not. A, Western blot analysis
showing expression of Sp1 protein (left panel) and Sp3
protein (right panel) in Sertoli cells. Each panel shows
whole cell extracts from Sertoli cells transfected with an irrelevant
plasmid (lanes 1), and whole cell extracts from Sertoli
cells transfected with the Sp1 or Sp3 expression vectors (lanes
2). B, Sertoli cells were co-transfected with the
FasL-Luc-3-wt or the FasL-Luc-3- Sp1 reporter gene constructs plus
1.5 µg of the Sp1 or the Sp3 expression vector, or both.
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To investigate the universality of the cis-acting Sp1 site in
controlling the basal transcriptional activity of the FasL gene, we
studied an unrelated cell line. Jurkat T cells were co-transfected with
the FasL-Luc-3 wt or FasL-Luc-3
Sp1 reporter constructs together or
not with Sp1 or Sp3 expression vectors and a TK-CAT to normalize for
transfection efficiency. As shown in Fig.
8, the basal transcriptional activity
from the FasL promoter is decreased by mutations in the Sp1 DNA binding
motif (columns 1 and 4). In addition, overexpression of Sp1
substantially increases transcription (column 2), while overexpression
of Sp3 increases transcription to a lesser degree (column 3).

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Fig. 8.
Basal FasL gene expression in Jurkat T cells
is regulated by Sp1. Unstimulated Jurkat T cells were
co-transfected with the FasL-Luc-3-wt or FasL-Luc3- Sp1 reporter gene
with Sp1 and/or Sp3 expression vectors.
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DISCUSSION |
We have demonstrated that an Sp1 consensus sequence is necessary
for basal transcription from the FasL promoter, and that Sp1 alone is
sufficient to drive transcription from the promoter in vivo.
The consensus sequence for Sp1 (GGGCGG) has been identified in the
promoter regions of a variety of genes although its role in gene
regulation is not understood (31). The site is known to bind Sp1, an
approximately 100 kDa, zinc-requiring transcription factor, and binding
results in increased transcription of the associated gene (32-34). Sp1
is essential for development in mice, as Sp1
/
embryos have
retarded growth and die early in gestation (35). Some studies have
suggested that Sp1 is involved in cell differentiation (36) as it has
been found in highest levels in hematopoetic stem cells, fetal cells,
and spermatids (35). Others have speculated that it is a major
transcription factor for housekeeping genes, as the Sp1 consensus
sequence is commonly found in the promoter region of these genes (37).
However, recent studies and our own data indicate that many mammalian
gene types are controlled by Sp1, including genes for structural
proteins, metabolic enzymes, cell cycle regulators, transcription
factors, growth factors, surface receptors, and others (31, 37). Our report adds the FasL gene to this growing list of relevant genes regulated by Sp1.
In Sertoli cells, Sp1 alone is sufficient to induce constitutive
transcription from the FasL promoter, while in several other systems
studied, Sp1 acts co-operatively with other transcription factors such
as AP-1 (38), E2F (39, 40), EGR1 (41, 42), and STAT 1 (43). In the
human immunodeficiency virus-long terminal repeat promoter region, Sp1
has been shown to interact with an NFKB site and controls basal
promoter activity (30). Although an imperfect NFKB site is present in
the FasL promoter, its significance for gene regulation in Sertoli
cells is unclear. Our data show that although mutations in this site
slightly decreased transcription from the FasL promoter, it is not due
to inhibition of NFKB binding to the site, and there appears to be no
significant functional interaction between Sp1 and NFKB.
We have shown that Sp3 is present in the protein complexes bound to the
Sp1 cis-acting motif in the FasL gene promoter, but it does not appear
to directly affect basal transcription in Sertoli or Jurkat T cells. In
other studies, Sp3 has been shown to function as either an inhibitor of
gene transcription (it can compete with Sp1 for binding to the
consensus sequence (44-47), or an activator (47). Sp3 activity is,
therefore, dependent on both the promoter characteristics and cellular
context. It will be interesting to understand the upstream regulation
of Sp1, and preliminary investigations have been done using other gene
systems. The data indicates that dysfunctions of Sp1 inhibitors result
in increased Sp1 activity and increased gene expression. Inhibitors
identified include Sp1 inhibitor (Sp1-I), which binds and inactivates
Sp1 (48-50), G10BP, which competes with Sp1 for binding to GC-rich
sequences in some promoters (44, 50, 51), and p107, which is a protein
related to the retinoblastoma protein that also binds and inactivates Sp1 (52). In addition, methylation of the Sp1 consensus site can
inhibit Sp1 binding (53). Inactivation of Sp3 and Sp1-I can occur by
the retinoblastoma protein (48-50), and Sp3 may also be inhibited by
IGF-1 in some systems (45).
Inducible FasL expression within T lymphocytes has been well
characterized and plays a major role in maintaining peripheral homeostasis of T cells by limiting the expansion of proliferating cells
during a normal immune response (5, 6). This process is mediated by the
transcription factor, NFAT, as previously shown by our group and others
(8, 10). Inducible FasL also functions as a cytolytic effector molecule
for cytotoxic T lymphocytes and NK cells (54, 55). However, the
significance of constitutive FasL expression by both immune and
non-immune cells is less well understood. It is known that constitutive
FasL expression by cells in immune-privileged sites and transplanted
grafts will protect these tissues from elimination by the immune
surveillance system (56, 57). The constitutive FasL expressed in
tissues not considered to be immune privileged may play a role in the
normal homeostasis of these tissues also (58, 59). Similarly, the
constitutive expression of FasL by macrophages may be involved in
controlling T cell homeostasis and play a role in human
immunodeficiency virus pathogenesis. Another area in which the role of
constitutive FasL expression is currently being addressed is that of
malignancy. Multiple types of malignancies constitutively express FasL
(17, 22, 23, 25, 27). While the functional relevance of this expression
has not been characterized, FasL may play a major role in the
invasiveness and metastatic capabilities of such tumors. Understanding
the constitutive transcriptional regulation of the FasL gene in such
tumors should help to identify specific transcription factors that are
relevant for understanding these malignant processes.
We have shown here that both the constitutive expression of FasL in
Sertoli cells and basal FasL transcription in T cell is controlled by
Sp1. Thus, it is tempting to propose that constitutive expression of
FasL in general may be controlled by Sp1, while inducible transcription
may be at least partially controlled by NFAT. This would be consistent
with prior data showing NFAT to be critical for the inducible
transcription of many other cytokine genes (60). Future investigations
of FasL gene transcription in additional cell types should clarify this issue.