©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nuclear Factor B Functions as a Negative Regulator for the Rat Androgen Receptor Gene and NF-B Activity Increases during the Age-dependent Desensitization of the Liver (*)

(Received for publication, May 25, 1994; and in revised form, October 17, 1994)

Prakash C. Supakar (§) Myeong H. Jung (§) Chung S. Song Bandana Chatterjee (¶) Arun K. Roy (**)

From the Department of Cellular & Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 kappaB (NF-kappaB) motif. Electrophoretic mobility shift analyses (EMSA) showed that the decameric rAR NF-kappaB site at positions -574 to -565 cross-competes with the authentic kappa immunoglobulin light chain enhancer for specific protein binding. Supershift with specific antibodies to NF-kappaB 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-kappaB/Rel proteins. Fragments of rAR promoter with either deletion or point mutation of the NF-kappaB 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-kappaB 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-kappaB 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-kappaB/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.


INTRODUCTION

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, (^1)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-kappaB/Rel belongs to a family of dimeric trans-acting factors, initially identified as an enhancer binding protein for the kappa immunoglobulin light chain in B lymphocytes (11) . Since then, a broader role of NF-kappaB 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-kappaB 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-kappaB/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.


MATERIALS AND METHODS

Preparation of Nuclear Extracts

Fischer 344 male rats of different ages were obtained from the colony of the NIA, National Institutes of Health, maintained at Charles River Breeding Laboratories. Ages of rats were accurate within ±10 days. All animal care and treatment protocols were approved by the Institutional Animal Care Committee. Animals were sacrificed and liver nuclear extracts were prepared according to the procedure described by Hattori et al.(17) .

Electrophoretic Mobility Shift Assay

Oligonucleotide probes used in this study are shown in Table 1. The upper strand of the duplex was end-labeled with [-P]ATP using T4 polynucleotide kinase. The labeled upper strand was annealed with a 5-fold excess of the unlabeled complementary strand. Nuclear extracts (5 µg) were preincubated in 20-µl reaction mixtures containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5-10% glycerol, and 1-2 µg of poly(dI-dC). After 5 min at room temperature, 10-20 fmol of P-end-labeled oligonucleotide duplex probe was added, and the incubation was continued for another 20 min(9, 18) . In competition experiments, the unlabeled competitor DNA (100-fold molar excess) was included during the preincubation period. Following electrophoresis, dried gels were either autoradiographed or analyzed in Betascope.



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-kappaB 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(2)O(2) treatment (150 µM, 60 min) were prepared as described(19) . Recombinant p50 protein produced in Escherichia coli was purchased from Promega Biotech.

DNase I Footprinting

The plasmid containing 530 bp of the AR promoter (-1040 to -511) was cleaved with KpnI and HindIII restriction endonucleases. The cleaved insert (573 bp) containing the 530-bp rAR promoter was labeled by end-filling with [alpha-P]dATP using Klenow DNA polymerase. Binding reactions were carried out using 50 µg of nuclear extract and 2 ng (30,000-60,000 cpm) of probe. For homologous competition, a 333-fold molar excess of the unlabeled oligonucleotide duplex was preincubated with the nuclear extract. Following DNase I digestion, DNA fragments were analyzed on a 6% polyacrylamide sequencing gel containing 7 M urea(9) .

Construction of Wild Type and Mutant Plasmids

The parental plasmids pAR (1.04)-Luc and pAR (0.665)-Luc used in this study contain the wild type rat androgen receptor promoter from positions -1040 to +19 and -665 to +19, respectively, inserted into the luciferase vector pGL2-Enhancer(5) . Deletion mutant as well as point mutant plasmids were used in this study. One mutant contains deletion at positions -574 to -554 within -665 bases of the AR promoter, pAR (0.665, Delta574/554)-Luc, and the second one has deletion at positions -574 to -565, pAR (0.665, Delta574/565)-Luc. The third mutant contains a base substitution at position -573, pAR (0.665, 573 G C). For a four-point AR mutant, the luciferase plasmid, containing -1040 bases of the AR upstream sequences, was mutated at 4 consecutive bases from -574 to -571 (GGGA ATCT) within the 10-bp NF-kappaB consensus sequence to create the mutant plasmid pAR (1.04, -574/-571 GGGA ATCT). Site-specific deletions were created by ligation of DNA fragments lacking the desired nucleotide residues. The DNA fragments of choice resulted from polymerase chain reaction (PCR) with appropriate primer pairs and the template DNA of the parental plasmid pAR (0.665)-Luc. For these two deletions, the ligated PCR fragments and corresponding primer pairs were as follows. Delta574/554: the 159-bp PCR fragment from the vector-specific GL1 primer and AR-specific antisense primer spanning positions -575 to -594 (5`-GACACCAAGCCATTTACTCT-3`) was ligated to a 653-bp DNA produced with the vector-specific GL2 primer and AR-specific sense primer spanning positions -553 to -531 (5`-CTGGAGGATCTCAAAGGTTTCTG-3`). Delta574/565: the same 159-bp DNA as above was ligated to a 664-bp PCR fragment from the vector-specific GL2 primer and AR-specific sense primer from -564 to -543 nucleotide residues (5`-CATCTACGCTACTGGAGGATCT-3`). The site-specific single point mutation was introduced initially on a DNA fragment generated by PCR using the plasmid template pAR (0.665)-Luc, in the presence of a mutant oligonucleotide primer spanning -609 to -561 positions containing a G C substitution at position -573 and a downstream vector-specific GL-2 primer. The four point mutations were introduced on a DNA fragment generated by PCR of the plasmid template pAR (0.665)-Luc in the presence of the upstream primer spanning -609 to -561 with 4 consecutive base substitutions from -574 to -571 (GGGA ATCT) and the downstream vector-specific GL2 primer. The mutant DNA fragments generated by PCR were digested with ApaI and purified by gel electrophoresis. Gel-purified primers were also used in all PCR experiments. The vector-specific GL1 and GL2 primers were from Promega Biotech, and the AR-specific primers were customsynthesized by the Midland Company. For the final construction of mutant plasmids, the wild type AR promoter fragments in the parental plasmids pAR (0.665)-Luc and pAR (1.04)-Luc were replaced by the mutant promoter fragments via the two ApaI sites at -604 and -459 positions of the rAR promoter sequence. All mutations were confirmed by nucleotide sequencing. Plasmid DNAs were purified through the Qiagen anion exchange resin, and plasmid concentrations were verified both by UV absorbance at 260 nm and by ethidium bromide staining following gel electrophoresis.

Cell Transfection and Enzyme Assay

Different cell lines used in this study were obtained from ATCC and grown in Dulbecco's modified Eagle's medium-Hank's F-12 medium (1:1, v/v) containing 10% fetal bovine serum. T flasks were seeded with 500,000 cells and cultured overnight before transfection. Test plasmids (10 µg) were transfected into cells by the calcium phosphate-DNA co-precipitation method(20) , and cells were harvested 24 h post-transfection. Cell extracts were assayed for luciferase activity (21) , and light emission was quantified in a Bio-Orbit 1250 luminometer. Protein concentrations of cell extracts were measured using the Bradford assay, and transfection results were computed as luciferase activities per mg of total protein.


RESULTS

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-kappaB and differs from the mouse immunoglobulin kappa chain enhancer (NF-kappaB site) at two positions (Table 1). However, these variant positions are not critical for specific binding of NF-kappaB to its cognate cis-element(22) . Additionally, this rAR element possesses a perfect dyad symmetry in contrast to other naturally occurring NF-kappaB motifs(14) .

Binding specificity of NF-kappaB 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 kappa 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 kappa oligonucleotide can be blocked with an excess of either the unlabeled rAR 21-mer oligonucleotide (lane 6) or the homologous NF-kappaB oligonucleotide (lane 7), but not with the mutant NF-kappaB 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 kappa enhancer. The labeled Ig kappa 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 kappa 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-kappaB, a 22-mer duplex corresponding to mouse Ig kappa light chain enhancer; and NF-kappaB, the mutant form of mouse Ig kappa 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-kappaB oligonucleotides, but not with the NF-kappaB mutant oligonucleotide. The two faster migrating bands produced only with the labeled NF-kappaB 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-kappaB 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 kappa 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-kappaB 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(2)O(2)-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-kappaB 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 kappa light chain enhancer (NF-kappaB). 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 kappa 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-kappaB during aging and after activation with phorbol ester and H(2)O(2). 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(2)O(2)-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-kappaB 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-kappaB deletion spanning positions -574 to -565 (665Delta10); (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 (665Delta21); (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-kappaB site (GGGA ATCT). Results in Fig. 4show that all four mutant promoters, with either base substitutions or deletions at the NF-kappaB 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-kappaB 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-kappaB-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-kappaB 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. GC, rAR promoter (up to -665) with a G C substitution at position -573 (n = 4); Delta10, rAR promoter (up to -665) with deletion of the 10-base pair palindromic NF-kappaB binding site (-574 to -565) (n = 3); Delta21, 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-kappaB in aging rats. Results presented in Fig. 5A demonstrate a gradual age-dependent increase in the NF-kappaB 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-kappaB binding activity as compared to the young adult (3-month-old) control. These results suggest that NF-kappaB may play an important role in the coordinated down-regulation of the rAR gene in the liver.

The hepatic NF-kappaB 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-kappaB 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-kappaB 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(2)O(2) (an agent that causes oxidative stress) markedly increased the nuclear level of the p50/p65 heterodimeric form of NF-kappaB (Fig. 5B). These results suggest a subtle difference between the pathways that mediate the age-dependent rise in the NF-kappaB activity and the acute inflammatory response.


DISCUSSION

The NF-kappaB/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-kappaB subunits possess different affinities for the cytoplasmic sequestering protein IkappaB which can be inactivated by phosphorylation (14) . Thus, the ultimate trans-regulatory effects of various dimeric species of NF-kappaB/Rel on a particular gene will depend on the cellular levels of the subunits and IkappaB, 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-kappaB/Rel dimers, the p50/p50 possesses the highest affinity for the decameric mouse kappa 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-kappaB. 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-kappaB/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-kappaB 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-kappaB elements and NF-kappaB expression vectors, Schmitz and Baeuerle (25) concluded that the homodimer of p50 has the potential of down-regulating kappaB-specific genes. A similar indication was also obtained in transgenic mice containing three copies of Ig kappa enhancer in front of a beta-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-kappaB/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-kappaB 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-kappaB 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-kappaB 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-kappaB 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-kappaB, ADF, and even some other as yet unidentified contributing factors.

Reactive oxygen intermediates serve as the major activating signal for NF-kappaB(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-kappaB can serve as the common signal carrier for both oxidative damage and inflammation and influence age-dependent dysfunctions.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants R-37 DK14744, RO1 AG10486, and PO1 AG06872 and a Veterans Administration Merit Review award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

Veterans Administration Career Scientist.

**
To whom correspondence should be addressed. Tel.: 210-567-3850; Fax: 210-567-3803.

(^1)
The abbreviations used are: ADF, age-dependent factor; rAR, rat androgen receptor; PCR, polymerase chain reaction; bp, base pair(s); EMSA, electrophoretic mobility shift assay; WT, wild type.


ACKNOWLEDGEMENTS

We thank our co-workers, Dr. Robert Vellanoweth and Dr. Kristina Detmer, for their interest and Tina Hassan and Sang Kim for technical assistance. Excellent secretarial help from Nyra White is highly appreciated.


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