(Received for publication, May 25, 1994; and in revised form, October 17, 1994)
From the
Transcriptional regulation of the steroid hormone receptor genes
plays a central role in temporal changes of target cell sensitivity
during development, maturation, and aging. Sequence-specific
DNA-protein interactions mediate these regulatory functions.
Progressive 5` deletion of the rat androgen receptor (rAR) gene
immediately beyond the -572 base pair (bp) region causes a marked
increase in its promoter activity. DNase I footprinting with nuclear
proteins revealed a protected area encompassing -574- to
-554-bp positions that begins with a perfectly palindromic
nuclear factor B (NF-
B) motif. Electrophoretic mobility shift
analyses (EMSA) showed that the decameric rAR NF-
B site at
positions -574 to -565 cross-competes with the authentic
immunoglobulin light chain enhancer for specific protein binding.
Supershift with specific antibodies to NF-
B subunits confirmed
that the two retarded bands observed in the EMSA with the labeled rAR
probe are due to p50/p65 and p50/p50 dimers of the NF-
B/Rel
proteins. Fragments of rAR promoter with either deletion or point
mutation of the NF-
B site are found to be about 2- to 3-fold more
effective as compared to the wild type control in driving a
heterologous reporter gene in cellulo. Thus, unlike most other
known cases, NF-
B acts as a negative regulator for the rAR gene.
The physiological relevance of this repressor function is evident from
a 10-fold increase in the p50/p50 form of the NF-
B activity in the
liver of aged rats exhibiting hepatic androgen desensitization. The
newly identified repressor element is a rare example of a naturally
occurring perfect palindromic binding motif for the NF-
B/Rel
family of transcription factors. This repressor factor and the
positively acting age-dependent factor, ADF, described earlier
(Supakar, P. C., Song, C. S., Jung, M. H., Slomczynska, M. A., Kim,
J.-M., Vellanoweth, R. L., Chatterjee, B. & Roy, A. K.(1993) J.
Biol. Chem. 268, 26400-26408) function to coordinate the
tissue-specific down-regulation of the rAR gene during aging.
The androgen receptor serves as a ligand-activated trans-acting factor for a large number of genes that are
directly and indirectly involved in the process of
reproduction(1, 2) . Tissue-specific regulation of
androgen sensitivity during development, maturation, and aging is
primarily mediated by selective expression of this receptor in target
cells. The promoter sequence of the androgen receptor gene from a
number of species, including man, has been
characterized(3, 4, 5) . A casual examination
of these sequences reveals binding sites for a variety of transcription
factors. In vitro DNA-protein interactions and transfection of
mutagenized promoters have established the roles of CREB, SP1, and a
novel transcription factor, ADF, ()in the regulation of the
androgen receptor gene(6, 7, 8, 9) .
Earlier studies in our laboratory showed that progressive 5` deletion
of the rat androgen receptor (rAR) gene immediately beyond the
-572-bp region resulted in a marked increase in the promoter
function(5) . This observation suggested that, in addition to
the positively acting factors mentioned above, a negative regulator may
be interacting around the -572 region of this gene. Because of
the age-dependent loss of hepatic androgen sensitivity, we were also
interested in exploring the possible role of this negative regulator in
the repression of the rAR gene activity during aging. The liver of the
male rat produces a number of androgen-induced proteins in relatively
large quantities, and the hepatic androgen sensitivity in this animal
declines gradually with age (10) . The steady state level of
the rAR mRNA in the liver of a young adult male rat (
3 months old)
is approximately 70-fold higher than that of a senescent animal
(
20 months old). The age-dependent nuclear factor (ADF), that
interacts with the rAR gene at the -329 to -311 site and
functions as a positively acting transregulator(9) , may only
account for about a 5-fold change in the rAR gene expression,
suggesting possible roles of other regulatory influences in the
age-dependent down-regulation.
NF-B/Rel belongs to a family of
dimeric trans-acting factors, initially identified as an
enhancer binding protein for the
immunoglobulin light chain in B
lymphocytes (11) . Since then, a broader role of NF-
B in
the regulation of many critical genes, especially those involved in
immune response, inflammation, oxidative stress, and embryonic
development, has been
appreciated(12, 13, 14, 15) .
NF-
B functions as either a homo- or heterodimer of a related group
of evolutionarily conserved proteins. The viral homolog of these
proteins acts as an oncogene(16) . Although the NF-
B/Rel
family of transcription factors generally mediates transcriptional
activation, here we present evidence for its negative regulatory
function that may play an important role in the age-dependent androgen
desensitization of the liver.
For antibody supershifts, 1 µl of the polyclonal rabbit
antiserum containing 0.05 µg of IgG was preincubated for 30 min at
22 °C with the nuclear extract in the absence of poly(dI-dC).
Subsequently, poly(dI-dC) was added and incubation continued for
another 5 min, followed by addition of the P-labeled probe
and further incubation for 20 min. Specific antibodies to NF-
B
subunits p50, p65, c-rel, and Rel B were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). HeLa nuclear extracts (derived
from control and phorbol ester-treated cells) were also obtained from
Santa Cruz Biotechnology. Jurkat cell nuclear extracts with and without
H
O
treatment (150 µM, 60 min) were
prepared as described(19) . Recombinant p50 protein produced in Escherichia coli was purchased from Promega Biotech.
Transfections of the progressively deleted rAR promoter have indicated that sequences immediately downstream of the -572 region may contain a negative regulatory element. In order to identify the existence of any specific protein binding site around this region, we have conducted DNase I footprinting analysis with liver nuclear extract and an end-labeled promoter fragment spanning the -1040 to -511 region of the rAR gene (Fig. 1). One of the footprints revealed in this experiment covered a 21-bp region encompassing positions -574 to -554. This protection is lost when a 333-fold molar excess of an oligonucleotide duplex corresponding to the -574 to -554 region was added as a competitor of the labeled DNA (Fig. 1, lane 4), substantiating the sequence specificity of the binding protein. Nuclear extracts from a number of heterologous sources including human (HeLa cells) were also effective in protecting the same footprint region (not shown in this figure).
Figure 1: DNase I footprint pattern of the rat androgen receptor (rAR) gene promoter spanning positions -1040 to -511 nucleotide residues. Lanes 1 and 2, naked DNA digested with two concentrations of DNase I (0.025 µg and 0.05 µg/ml). Lane 3, DNA treated with 0.25 µg/ml DNase I in the presence of 50 µg of rat liver nuclear extract (RLNE). Lane 4, reaction conditions are the same as those of lane 3, except that a 333-fold molar excess of a 21-bp-long oligonucleotide duplex corresponding to positions -574 to -554 nucleotides of the rAR promoter was added to the reaction before digestion. Lane 5, G + A sequencing ladder of the same DNA fragment used for DNase I digestion. The nucleotide sequence of the protected region (-574 to -554) is presented on the right.
A computer search of the transcription factor
data base revealed that the 5` half of the 21-bp protected region shown
in Fig. 1conforms to the 10-base pair consensus binding
sequence of NF-B and differs from the mouse immunoglobulin
chain enhancer (NF-
B site) at two positions (Table 1).
However, these variant positions are not critical for specific binding
of NF-
B to its cognate cis-element(22) .
Additionally, this rAR element possesses a perfect dyad symmetry in
contrast to other naturally occurring NF-
B motifs(14) .
Binding specificity of NF-B to the 21-bp protected region
identified by DNase I footprinting was further characterized by
electrophoretic mobility shift analysis (EMSA). A labeled
oligonucleotide duplex corresponding to the footprinted site produced
two retarded bands (Fig. 2). Both of these retarded bands could
be competed with a 100-fold molar excess of either the unlabeled
homologous oligonucleotide duplex (21-mer) or with a 22-mer
oligonucleotide containing the wild type mouse immunoglobulin
chain enhancer element (lanes 2 and 3). However, the
mutant oligonucleotide with a critical G
C substitution (Table 1) failed to compete with the labeled probe for protein
binding (lane 4). Furthermore, specific protein binding of the
labeled mouse Ig
oligonucleotide can be blocked with an excess of
either the unlabeled rAR 21-mer oligonucleotide (lane 6) or
the homologous NF-
B oligonucleotide (lane 7), but not
with the mutant NF-
B oligonucleotide. These results indicate that
in the two retarded bands, the proteins binding to the labeled rAR
oligonucleotide have binding specificity similar to those binding to
the mouse Ig
enhancer. The labeled Ig
oligonucleotide
produced two additional retarded bands (marked with asterisks), and binding was substantially reduced by
competition with the homologous oligonucleotide (lane 7), but
not with an excess of the rAR oligonucleotide (lane 6). Since
the rAR oligonucleotide did not generate these two additional bands, we
have not explored them further. The two slower migrating specific bands
produced with the labeled rAR oligonucleotide (-574/-554)
can also be eliminated by excess truncated 12-mer oligonucleotide
duplex corresponding to positions -574 to -563 of the rAR
gene (not shown in this figure).
Figure 2:
Cross-competition between the rat androgen
receptor -574 to -554 oligonucleotide and the
immunoglobulin chain enhancer for specific protein binding.
Electrophoretic mobility shift analyses were carried out with the
labeled oligonucleotide as indicated on the top of the figure.
Unlabeled competing oligonucleotide duplexes (100-fold molar excess)
identified on the figure in abbreviated forms are: -574/-554, a 21-mer duplex corresponding to
-574 to -554 region of rAR; NF-
B, a 22-mer
duplex corresponding to mouse Ig
light chain enhancer; and NF-
B
, the mutant form of mouse Ig
light chain enhancer with a critical G to C substitution. All of
the sequence information is provided in Table 1. The two retarded
bands on the top of the gel showed cross-competition with the
rAR and NF-
B oligonucleotides, but not with the NF-
B mutant
oligonucleotide. The two faster migrating bands produced only with the
labeled NF-
B oligonucleotide (marked with asterisks) did
not compete with the rAR oligonucleotide.
Identity of the nuclear factors
that interact with this particular region of the rAR promoter was
further authenticated by antibody supershift experiments. Results
presented in Fig. 3show that the antibody which is specific for
the p50 subunit of NF-B supershifted all of the faster migrating
lower band and removed some of the upper complex (lane 2). The
antibody specific for p65, on the other hand, selectively reduced the
slower migrating upper band (lane 3). Antibodies to c-rel and Rel B did not have any effect on either of these two bands (lanes 4 and 5). Similar reactivity to antibodies
against p50 and p65 was also observed when an oligonucleotide
containing the mouse Ig
enhancer was used as the labeled probe (lanes 7-10). These results, in conjunction with
observations of Fujita et al.(23) with respect to
relative electrophoretic mobilities of protein-DNA complexes of various
recombinantly produced NF-
B subunits, allow us to conclude that
the faster migrating retarded band may contain a homodimer of p50 while
the slower migrating upper band is most likely due to the binding of a
p50/p65 heterodimer. Selective removal of the upper band with the p65
antibody rather than its supershifting may be due to the formation of a
microprecipitate in the reaction tube. Conclusions concerning the
identities of the two retarded bands were also substantiated by
observations that (i) the protein-DNA complex generated by the
recombinantly produced p50 and the labeled -574/-554 rAR
oligonucleotide co-migrates with the lower band (not shown here) and
(ii) a large increase of the upper band was observed for the
H
O
-treated Jurkat cell nuclear extract that is
known to contain a highly induced level of the p50/p65 heterodimer
(shown in Fig. 5)(19, 22) .
Figure 3:
Immunoreactivity of the androgen receptor
promoter binding protein with antibodies to NF-B subunits.
Electrophoretic mobility shift assays were performed with labeled
oligonucleotides with sequences corresponding to either -574 to
-554 (-574/-554) segment of the rAR gene or the mouse
immunoglobulin
light chain enhancer (NF-
B). Specific
antibodies were added to individual samples as indicated on the top of the panel. The slowest migrating band is removed by
the antibody to p65 (lanes 3 and 8). The band
immediately below the slowest migrating band is supershifted with the
antibody to p50 (lanes 2 and 7). The faster migrating
two additional bands produced with the immunoglobulin
chain
oligonucleotide (lanes 6-10) did not show any
immunoreactivity (marked with asterisks).
Figure 5:
Differential increase in p50/p50 and
p50/p65 forms of NF-B during aging and after activation with
phorbol ester and H
O
. A, liver nuclear
extracts derived from animals of different ages showing a gradual and
preferential increase in the p50/p50 homodimer activity. B,
preferential increase in p50/p65 heterodimer activity in PMA-treated
HeLa cells (lanes 1 and 2) and in
H
O
-treated Jurkat cells (lanes 3 and 4). For both A and B, rAR
-574/-554 oligonucleotide duplex was used as the labeled
probe. In order to maintain a visible separation between p50/p50 and
p50/p65 complexes, even at elevated concentrations, the x-ray films
were intentionally underexposed.
Having
established the authenticity of the NF-B binding site at the
upstream region of the rAR gene, we wanted to examine its regulatory
function. DNA constructs containing different versions of the rAR gene
promoter ligated to the firefly luciferase structural gene were
transfected into CHO cells. Promoters used for these experiments were
as follows: (i) the wild type rAR promoter containing up to a
-665-bp segment of the gene (665 WT), (ii) a point mutant of the
665 WT containing G
C substitution at position -573 (665 G
C); (iii) a deletion mutant that contained the wild type
sequence up to -665 with a 10-bp NF-
B deletion spanning
positions -574 to -565 (665
10); (iv) a deletion mutant
that contained upstream sequences up to -665, but lacked the
entire 21-bp DNase I protected region spanning positions -574 to
-554 (665
21); (v) a longer promoter segment containing up to
-1040 bp in its wild type form (1040 wt); and (vi) a mutant form
of the -1040-bp promoter (1040:4 Pmt) containing 4 base
substitutions within the NF-
B site (GGGA
ATCT). Results in Fig. 4show that all four mutant promoters, with either base
substitutions or deletions at the NF-
B site, were approximately
2-3-fold more effective in driving the expression of the
luciferase reporter gene compared to the wild type control. From these
results and the promoter activities of deletion mutants described
before(5) , we conclude that the NF-
B site on the rAR
promoter functions as a negative regulatory element in transfected
cells. We have repeated these experiments with other cell lines besides
CHO, including COS-1 and HepG2 cells. With these cells, although the
extent of increase of the NF-
B-deleted promoter varied to some
extent, in all cases the mutant promoters were more effective in
driving the reporter gene as compared to the wild type control.
Figure 4:
Biological activity of the rat androgen
receptor gene promoter with mutations at the NF-B binding site in
CHO cells. The wild type (WT) promoter-reporter constructs contained
+19 to -665 bases (hatched bar) and +19 to
-1040 bases (open bar) of the rAR gene and the firefly
luciferase structural gene. Luciferase activities with mutant promoter
constructs are expressed as percent of the wild type ± S.E. G
C, rAR promoter (up to -665)
with a G
C substitution at position -573 (n = 4);
10, rAR promoter (up to -665)
with deletion of the 10-base pair palindromic NF-
B binding site
(-574 to -565) (n = 3);
21,
rAR promoter (up to -665) with a deletion of the whole 21-bp
footprint site (-574 to -554) (n = 3); 4Pmt, rAR promoter (up to -1040) with 4 base
substitutions from positions -574 to -571 (n = 3). The number of independent transfections is indicated
by n. For each plasmid, at least two different batches of
plasmid preparation were used in transfection. For each independent
batch of transfection assay, values were averages of duplicate (for G
C mutant) or triplicate (for the other three mutant plasmids)
transfections.
The
physiological relevance of these in vitro studies to the
age-dependent decline in rAR gene expression was explored by examining
the hepatic levels of NF-B in aging rats. Results presented in Fig. 5A demonstrate a gradual age-dependent increase in the
NF-
B binding activity in the liver. The nuclear extract from the
liver of a 26-month-old senescent male rat which shows a markedly
reduced AR gene expression(9) , contains an approximately
10-fold higher NF-
B binding activity as compared to the young
adult (3-month-old) control. These results suggest that NF-
B may
play an important role in the coordinated down-regulation of the rAR
gene in the liver.
The hepatic NF-B activity increases under
conditions of inflammation, and older animals are known to be more
susceptible to chronic inflammatory responses relative to their younger
counterparts. Furthermore, acute inflammatory response preferentially
increases the p50/p65 heterodimeric form of NF-
B over the p50/p50
homodimer(19, 22) . It was, therefore, of interest to
explore any possible difference between the age-dependent increase in
the hepatic NF-
B activity and an acute inflammatory response.
Results presented in Fig. 5A show that aging causes a
preferential increase in the hepatic level of the p50/p50 homodimer.
However, as expected, both HeLa cells treated with phorbol myristic
acetate (an activator of protein kinase C and inflammatory response)
and Jurkat T cells treated with H
O
(an agent
that causes oxidative stress) markedly increased the nuclear level of
the p50/p65 heterodimeric form of NF-
B (Fig. 5B).
These results suggest a subtle difference between the pathways that
mediate the age-dependent rise in the NF-
B activity and the acute
inflammatory response.
The NF-B/Rel family constitutes a group of dimeric
transcriptional regulators containing an approximately 300-amino acid
long highly conserved Rel homology (RH) domain. About 12 different
dimeric combinations of the five well-characterized subunits have been
implicated in the transcriptional regulation of a number of genes that,
among others, are involved in immune response, inflammation, acute
phase response, and embryonic differentiation(14) . It appears
that the homologous RH domain provides both the DNA binding and
dimerization sites, and the nonhomologous segments of the component
subunits along with the overall dimeric protein conformation may
primarily be responsible for the transcriptional
regulation(23) . Individual members of NF-
B subunits
possess different affinities for the cytoplasmic sequestering protein
I
B which can be inactivated by phosphorylation (14) .
Thus, the ultimate trans-regulatory effects of various dimeric species
of NF-
B/Rel on a particular gene will depend on the cellular
levels of the subunits and I
B, the binding affinity of the
response element for the available dimeric forms, the intrinsic
subunit-specific transregulatory capacity, and the interaction with
other non-Rel proteins functioning as co-activators or co-repressors.
Earlier studies of Fujita et al.(23) have shown
that among various NF-B/Rel dimers, the p50/p50 possesses the
highest affinity for the decameric mouse
immunoglobulin light
chain response element while p50/p65, a weaker binder, provides
stronger transactivation. Furthermore, the differential DNA binding
ability of p50/p50 over p50/p65 increases as the dyad symmetry of the
binding site nears perfection(23, 24) . In this
article we report evidence for a naturally occurring perfectly
palindromic binding site for NF-
B. Judging from the results of
Fujita et al.(23) , this rAR element will possess
anywhere between 5- and 10-fold higher affinity for p50/p50 than its
more potent trans-activating homolog p50/p65. Since p50/p50
and p50/p65 appear to be the predominant forms of NF-
B/Rel in
liver nuclear extracts, in the absence of other determining factors,
the p50/p50 homodimer may preferentially occupy the rAR element in
vivo. In aging animals, such a preferential occupancy of the
NF-
B site on the rAR promoter will be further ensured by the
age-dependent increase in the p50/p50 homodimer.
Despite its
generally recognized transactivating function, several lines of
evidence have already suggested the potential repressor role of the
p50/p50 homodimer. Based on the results of co-transfection, experiments
with a basal promoter containing multimerized NF-B elements and
NF-
B expression vectors, Schmitz and Baeuerle (25) concluded that the homodimer of p50 has the potential of
down-regulating
B-specific genes. A similar indication was also
obtained in transgenic mice containing three copies of Ig
enhancer in front of a
-globin reporter gene(26) . In the
latter case, the authors observed that organs that only contain the p50
homodimer failed to express the transgene. More recently, Brown et
al.(27) have provided evidence for the possible role of
p50 homodimer in the cell-specific down-regulation of the major
histocompatibility complex class II-associated invariant chain mRNA.
However, the mechanism of the inhibitory function of the p50/p50
homodimer at this point remains speculative. It is possible that the
repressor function may require the help of some other DNA binding
protein that can act as a co-repressor. The Drosophila homolog
of the NF-
B/Rel family, known as dorsal (dl),
simultaneously functions as an activator for regulatory genes such as twist and snail and as a repressor for genes such as decapentaplegic (dpp) and Zerknütt (Zen)(15) . Genes
that are negatively regulated by dl contain a minimal 110-bp
ventral response element. In addition to two dl binding sites,
the ventral response element contains several co-repressor binding
sites that are essential for repression. Mutations in some of these
co-repressor binding sites convert the minimal ventral response element
into an activator element. Such a mechanism may also be possible for
the p50/p50-mediated repression of the rAR gene. If a co-repressor,
bound to a noncontiguous site at the 3` end of the NF-
B site,
interacts with the p50/p50 homodimer through DNA looping and
protein-protein interaction, that may also explain the 11-base pair 3`
extension of the footprint site beyond the decameric NF-
B site on
the rAR gene. It is noteworthy that band shift with oligonucleotide
duplexes containing the entire footprint region did not produce any
additional retarded bands other than the NF-
B complexes,
suggesting that proteins that confer the extended protection may
require downstream DNA sequences for stabilization.
The
age-dependent decline in rAR gene expression appears to be coordinated
by more than one trans-regulator. In addition to the negative
regulatory influence of NF-B and the increase of the p50/p50
binding activity during aging, we have recently described the positive
regulatory role of a novel age-dependent factor (ADF) in the
down-regulation of the rAR gene(9) . ADF is an evolutionarily
conserved transcription factor whose activity in the liver nuclear
extract declines about 7-fold during aging (from 3 months to 26
months). Deletion or point mutation of the ADF binding site on the rAR
gene promoter causes a 5-fold decline in the promoter activity in
transfected cells. However, an approximate 70-fold decline in the
steady state level of rAR mRNAs in the liver of senescent rats relative
to the young adult control may require the coordinated action of
NF-
B, ADF, and even some other as yet unidentified contributing
factors.
Reactive oxygen intermediates serve as the major activating
signal for NF-B(19, 22) . The hypothesis that a
cumulative cellular damage produced by oxygen radicals is one of the
major driving forces for aging (28) has recently been
strengthened by the observation that simultaneous overexpression of
superoxide dismutase and catalase in transgenic flies lengthens their
life span(29) . A progressive dysregulation of the inflammatory
response also appears to be one of the phenotypic changes associated
with aging(30) . It is therefore reasonable to assume that the
ubiquitous transregulator NF-
B can serve as the common signal
carrier for both oxidative damage and inflammation and influence
age-dependent dysfunctions.