(Received for publication, January 26, 1996)
From the
A DNA response element, TR2RE-EPO (5`-TCTGACCTCTCGACCTAC-3`) has
been identified in the 3`-minimal hypoxia-inducible enhancer of the
human erythropoietin gene for the TR2 orphan receptor, an
androgen-repressed transcription factor and a member of the
steroid/thyroid hormone receptor superfamily. Electrophoretic mobility
shift assay showed a specific binding with high affinity (K = 0.14 nM) between the
TR2 orphan receptor and the TR2RE-EPO. Our data further indicated that
this specific binding is not due to the homo-dimerization of the TR2
orphan receptor. In addition, reporter gene expression using
chloramphenicol acetyltransferase assay demonstrated that the TR2
orphan receptor may suppress the expression of the chloramphenicol
acetyltransferase activities via the TR2RE-EPO in the hypoxic/normoxic
human hepatoma HepG2 cells. Finally, our in situ hybridization
data also indicated that the TR2 orphan receptor and the erythropoietin
transcripts can be co-expressed in mouse kidney and liver. Together,
our data suggest that the human erythropoietin gene could represent the
first human target gene regulated directly by the human TR2 orphan
receptor.
Members of the steroid/thyroid hormone receptor superfamily are
transcriptional factors that regulate the expression of target genes by
binding to specific cis-acting sequences in the nuclei of
animal cells(1) . These nuclear receptors include receptors for
steroid, thyroid, vitamin D, vitamin A-derived hormones,
and a large number of orphan receptors in which cognate ligands have
not yet been identified(2, 3) . Numerous orphan
receptors have been identified by low stringency hybridization
screening and other cloning techniques (4, 5) (reviewed in (6) and (7) ).
Thus, they share common modular architecture within the superfamily,
including a variable N-terminal portion, a high degree of homology in
the DNA-binding domain with two zinc fingers, and a putative
ligand-binding domain at the C-terminal region. Consequently,
physiological roles of orphan receptors have been postulated and
subjected to speculation since they were initially identified. More
recent efforts exploring their potential functions have demonstrated
the remarkable impact of the nuclear receptor superfamily. Novel
ligands or activators for several orphan receptors have been
identified, for instance, 9-cis-retinoic acid,
15-deoxy-
-prostaglandin J
, and
melatonin (5-methyoxy-N-acetyltryptamine) for retinoid X
receptor (RXR), (
)peroxisome proliferator-activated receptor
, and retinoid Z receptors
and
(8, 9, 10, 11) . In addition,
some orphan receptors are constitutive transactivators or repressors,
such as the TR3 orphan receptor or COUP-TF
I(12, 13, 14, 15, 16, 17) .
Certain orphan receptors and classical steroid receptors can be
activated to regulate gene transcription by modulators, such as
neurotransmitters (dopamine), or by internal changes in phosphorylation
pathways(6) . Several orphan receptors, however, may function
as co-regulators of ligand-dependent receptors to modulate
ligand-mediated signaling pathways at the protein or DNA level; for
example, RXR heterodimerizes with respective receptors for retinoic
acid (RAR), thyroid hormone, peroxisome proliferator-activated, and
vitamin D
(18) .
The human TR2 orphan receptor is
one of the first orphan receptor identified that shares structural
homology with members of the steroid/thyroid hormone receptor
superfamily(4, 19) . The TR2 orphan receptor cDNAs
were isolated from both human prostate and testis cDNA libraries using
a probe homologous to a highly conserved DNA-binding domain common to
steroid hormone receptors. The TR2-11 orphan receptor cDNA
encodes a protein of 603 amino acid residues with a calculated
molecular mass of 67 kDa. We also identified a distinct set of cDNAs,
named the human TR4 orphan receptor, from human prostate and testis
cDNA libraries(20) . The amino acid sequence of the TR4 orphan
receptor is closely related to that of the TR2 orphan receptor. This
high homology between the TR2 and TR4 orphan receptors highlights a
unique subclass within the steroid/thyroid hormone receptor
superfamily. Northern blot analysis showed that the TR4 orphan receptor
could be detected in many tissues in humans and
mice(20, 21) . The expression of the TR4 orphan
receptor transcripts in the human kidney is significantly more than
that in the liver. Recently, the rat TR2 orphan receptor cDNAs encoding
590 amino acids were isolated from rat prostate cDNA
library(22) . In addition, the genomic locus of the TR2 orphan
receptor gene has been mapped to the human chromosome 12q22. ()More recently, we demonstrated that the TR2 orphan
receptor may modulate the activation of both RAR and RXR hormone
response elements (HREs). This suggested that the TR2 orphan receptor
may be a master regulator in the retinoic acid signal transduction
pathway(23) . Moreover, we have identified a TR2 orphan
receptor response element (TR2RE-SV40) in the transcriptional
initiation site of the SV40 major late promoter(24) . This DNA
response element contains a direct repeat of AGGTCA consensus motif,
and the TR2 orphan receptor may function as a repressor for SV40 gene
expression.
Erythropoietin (EPO) is an essential survival and growth factor for the erythrocytic progenitor cells in the bone marrow (reviewed in (25, 26, 27) ). This glycoprotein hormone containing 165 amino acids, with a molecular mass of 30.4 kDa, is synthesized mainly in the kidney and fetal liver in response to hypoxia in mammals(28, 29, 30) . The mechanism for the induction of the EPO gene expression by the lack of oxygen is only partially understood. EPO deficiency is the primary cause of anemia in chronic renal failure. The human EPO gene has been cloned and expressed in vitro in mammalian cell cultures(31, 32) . The cis-acting elements of the human EPO gene responsible for hypoxic induction were identified in both the 5`-promoter and 3`-flanking regions(33, 34, 35, 36) . This 3`-enhancer is a highly conserved region located 120 base pairs (bp) downstream of the polyadenylation site among several species, and contributes a 4-14-fold induction of reporter gene expression in a wide variety of cell lines(37) . Recent studies have further narrowed down this enhancer to a 50-bp element consisting of at least three transcriptional factor binding sites(38) . The first site, a highly conserved 9-bp near the 5`-end of the minimal enhancer, can be bound by a 120-kDa hypoxia-inducible factor from nuclear extracts. The second one, containing three CA repeats, has not yet been characterized. However, the last one, located at the 3`-end of the minimal enhancer, consists of a direct repeat of AGGTCA consensus motif separated by 2 bp(35) . This site has been suggested as a potential DNA response element for a few orphan receptors, such as the hepatic nuclear factor 4, COUP-TF I, and TR2 orphan receptors(39) . We are interested in knowing if the TR2 orphan receptor may bind specifically to this EPO enhancer and play a role in the control of EPO gene expression. In the present study, we developed poly- and monoclonal anti-TR2 orphan receptor antibodies as probes to explore the possible consequences of this specific binding between the TR2 orphan receptor and the EPO enhancer. Our data demonstrate that the TR2 orphan receptor may function as a repressor in EPO gene regulation. Thus, the EPO gene could represent the first identified human target gene regulated by the TR2 orphan receptor.
The pET system is a powerful
system to express recombinant proteins in E.
coli(42) . The expression of the TR2 orphan receptor from
the pET system was performed according to the manufacturer's
instruction (Novagen). Basically, plasmid pET-TR2 containing the
DNA-binding domain of the TR2 orphan receptor cDNA with six consecutive
histidine residues at the N terminus was transformed into the
BL21(DE3)pLysS host strain (Novagen), and cultured in NZCYM (10 g NZ
amine, 5 g NaCl, 5 g yeast extract, 1 g casamino acids, 2 g
MgSO7H
O (pH 7.5) per liter) medium until
the OD
reached 0.6 with shaking at 37 °C. Isopropyl
-D-thiogalactopyranoside was added to a final
concentration of 0.5 mM, and the incubation was continued for
an additional 3 h. E. coli were harvested by centrifugation at
7,000 rpm for 5 min at 4 °C, and resuspended in one-fourth of the
culture volume of the binding buffer (40 mM Tris-HCl (pH 7.9),
5 mM imidazole, and 0.5 M NaCl). Bacteria were lysed
through one freeze/thaw cycle followed by homogenization (OMNI 2000,
Omni International). The lysates were centrifuged at 3,000 rpm (Beckman
GPR) for 20 min at 4 °C. The cellular extractions were then either
analyzed on SDS-polyacrylamide gel electrophoresis followed by
Coomassie Blue staining, or purified by a one-step metal chelation
chromatography (Novagen). For the production of polyclonal antibody,
the purified peptide was emulsified with Freund's complete
adjuvant and injected intradermally into New Zealand White rabbits
(Medical School Animal Care Unit, University of Wisconsin-Madison) as
described previously(41) .
In situ hybridization was performed mainly as described elsewhere. ()Briefly, mouse embryo sections were deparaffinized,
hydrated, and then treated with proteinase K (Boehringer Mannheim) at
20 µg/ml for 7.5 min. After washing in phosphate-buffered saline,
the sections were fixed in 4% paraformaldehyde, acetylated, dehydrated,
and air-dried. These sections were hybridized at 52 °C for 17 h
with cRNA probes (the specific activity for both sense and antisense
probes reached 1
10
cpm/µg). The probe
(10
cpm) was included in the hybridization buffer
containing 50% formamide for each slide. Washes were performed with
high stringency (2
SSC, 50% formamide at 65 °C (1
SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH
7.0)) before and after RNase digestion (20 µg/ml for 30 min).
Slides were dipped into Kodak NTB2 emulsion and exposed for 6 weeks.
Subsequently, slides were developed in Kodak D19 developer, fixed,
dehydrated, and mounted for light-field analysis.
Figure 1:
Construction of the recombinant TR2
orphan receptor baculovirus expression plasmid and Western blot
analysis with anti-TR2 orphan receptor monoclonal antibody. A,
construction of the recombinant TR2 orphan receptor baculovirus
expression plasmid. The full-length coding region of the TR2 orphan
receptor cDNA was cloned under the control of the polyhedrin promoter
in the pVL1393 baculovirus transfer vector as detailed under
``Materials and Methods.'' B, Western blot analysis
with anti-TR2 orphan receptor monoclonal antibody. Cellular extract
proteins (10 µl from a 1 10
cell/ml lysate
solution) from S. frugiperda 9 insect cells (lane 1),
insect cells infected with the wild-type baculovirus (lane 2),
the recombinant TR2-baculovirus (lane 3), or the purified TR2
orphan receptor from the recombinant TR2-baculovirus infected lysate (lane 4) were analyzed on a 4-20% gradient gel on
SDS-polyacrylamide gel electrophoresis, blotted to an Immobilon-P
membrane, and detected with the monoclonal anti-TR2 orphan receptor
antibody G204-218.48. Positions of molecular mass markers (kDa) are
indicated on the left; the TR2 orphan receptor is indicated by
the arrowhead.
Figure 2:
Binding of the in vitro expressed
TR2 orphan receptor to the 3`-EPO enhancer region. EMSA was performed
with the in vitro expressed TR2 orphan receptor and the P-end-labeled DNA probe. Lane 1 displays the
probe alone, which contains the 50-bp minimal hypoxia-inducible
enhancer(38) . Binding reaction mixtures incubated with the
probe and either mock-translated product (lane 2) or the in vitro synthesized TR2 orphan receptor (lanes
3-7) in the presence of unlabeled oligonucleotides (lane
4), preimmunized serum (preim, lane 5),
polyclonal anti-TR2 orphan receptor antibody #1132 (poly, lane 6), or monoclonal anti-TR2 orphan receptor antibody
G163-23 (mono, lane 7) are shown. The retarded
complexes are indicated by the arrowhead for specific
DNA-protein complexes, whereas the supershift band is marked by the arrow for DNA-protein-antibody
complexes.
To determine the DNA-protein binding
affinity between the TR2 orphan receptor and the EPO enhancer, we
performed Scatchard binding analysis by the EMSA. As shown in Fig. 3, constant amounts of the TR2 orphan receptor (60 ng) were
incubated with different concentrations of the DNA probe
(0.4-12.8 ng). DNA-protein complexes were resolved in the EMSA (Fig. 3A). Scatchard plot analysis resulted in a single
binding component for the specific DNA-protein complex with a
dissociation constant (K) of 0.14 nM and B
of 0.005 nM (Fig. 3B). These results fit well into the range
of K
for classical steroid receptors and their
HREs.
Figure 3:
Binding affinity of the TR2 orphan
receptor to the 3`-EPO enhancer. A, binding of the in
vitro expressed TR2 orphan receptor to various concentrations of
the probe in the EMSA. Constant amounts of the in vitro expressed TR2 orphan receptor (60 ng) were incubated with
different concentrations of the probe (0.4-12.8 ng). The specific
DNA-protein complex (indicated by the arrowhead) and the free
probe at the bottom were quantified by PhosphorImager (Molecular
Dynamics). Six points of experimental data are shown here. B,
Scatchard plot analysis. The ratio between specific DNA-protein binding (bound, nM) and free DNA probe with
respect to specific DNA-protein binding (bound/free) was
plotted. The dissociation constant (K)
and B
values were generated from Ebda software
(Biosoft).
Figure 4:
Domain architecture of the TR2 orphan
receptor in the recognition of the 3`-EPO enhancer. A,
schematic structure of various truncations of the TR2 orphan receptor.
Plasmid pSG5-TR2 contains the full-length TR2 orphan receptor coding
region, whereas plasmids pSG5-STR2, pSG5-3STR2, and pSG5-CTR2 represent
two C-terminal and one N-terminal truncations of the TR2 orphan
receptor, respectively. The DNA-binding domain (DBD) is
included in these constructs. Each number shows the amino acid residue
number within the TR2 orphan receptor cDNA. Molecular masses of the
intact TR2 orphan receptor, two C-terminal, and one N-terminal
truncations of the TR2 orphan receptors are indicated. B,
analysis of the in vitro expressed TR2 orphan receptor and its
variants in SDS-12% polyacrylamide gel electrophoresis. Lanes 1 and 6 show C-labeled methylated protein
standards and mock-translated products, respectively. Lanes
2-5 display the intact TR2 orphan receptor, two C-terminal,
and one N-terminal truncations, respectively. C, binding of
the binary mixture of the TR2 orphan receptor and its variants to the
EPO enhancer. Lane 1 displays the DNA probe alone. Binding
reaction mixtures incubated with the probe and mock-translated product (lane 2), the intact TR2 orphan receptor (lanes 3-5 and 15-23), the extreme C-terminal truncated TR2
orphan receptor (lanes 6-8 and 15-17),
the C-terminal deleted TR2 orphan receptor (lane 9-11 and 18-20), or the N-terminal truncated TR2 orphan
receptor (lanes 12-14 and 21-23), in the
presence of unlabeled oligonucleotides (lanes 4, 7, 10, 13, 16, 19, and 22),
or polyclonal anti-TR2 orphan receptor antibody (lanes 5, 8, 11, 14, 17, 20, and 23) are shown. The retarded complexes are indicated by small, medium, and large arrowheads for the
DNA-truncated TR2 orphan receptor complexes, the DNA-intact TR2 orphan
receptor complexes, and DNA-protein-antibody complexes,
respectively.
Figure 5:
The TR2 orphan receptor represses the EPO
gene expression by the CAT assay. Human hepatoma HepG2 cells were
co-transfected with expression vectors containing either the
full-length TR2 orphan receptor expression plasmid (pSG5-TR2, lanes
2 and 5) or the chimeric TR2 orphan receptor expression
plasmid (pSG5-TR2/ARp/TR2, lanes 3 and 6), and
reporter plasmids consisting of either the parent reporter
pCAT-promoter plasmid (lanes 1-3) or pSVcatEJ plasmid (lanes 4-6) in normoxia (open bar) or hypoxia (closed bar). Plasmid pSVcatEJ contains a 50-bp fragment of
the minimal hypoxia-inducible enhancer in the pCAT-promoter
vector(38) . All CAT assays were standardized for the level of
-galactosidase activity. The normalized ratio of relative CAT
activity at hypoxia (100 µM of CoCl
) or
normoxia is shown. Each value represents the average of at least three
independent experiments with the error bar designating
standard deviation.
Figure 6:
Localization of the TR2 orphan receptor
transcripts in the mouse embryo. All in situ hybridization
experiments were performed by the S-UTP-labeled antisense
mouse TR2 orphan receptor riboprobes. Exposure time was 6 weeks for all
slides. Photomicrographs of autoradiograms are presented for sagittal
sections of mouse embryos at 14.5 dpc (A) and 16.5 dpc (B-D). Tissues and organs with strong hybridization signals
(dark areas in bright field) are labeled, for example, the brain (b). C, photoemulsion-dipped sections at higher
magnification showed strong signals within the kidney (k) but
weak within the liver (li), while the pancreas (p)
served as a negative control. D, magnified sections in the
kidney revealed signals in the developing glomeruli (gl),
proximal tubules (pt), and interstitium between tubules (int). The bars represent 1 and 0.5 mm of length in panels A-C and D,
respectively.
The mechanism for hypoxia or cobalt chloride triggering the increased production of the EPO mRNA and protein remains one of the major unsolved mysteries in EPO gene regulation(26, 30) . In this study, we have demonstrated that the human TR2 orphan receptor may suppress the human EPO gene expression via the 3`-transcriptional enhancer. This implies that the TR2 orphan receptor may function as a negative modulator in EPO gene regulation. It is interesting to note that androgenic steroids have been shown to increase the production of EPO in mammals(51, 52) . Prior to the introduction of recombinant human EPO in 1985, androgenic therapy was widely used by clinicians, and is still used occasionally(53) . More recently, clinical trials have shown that replacement therapy with recombinant human EPO can benefit patients with anemia of chronic renal failure, myelodysplastic syndrome, acquired immunodeficiency syndrome, hemoglobinopathies, or malignancies(26, 27, 53) . However, not all patients respond to or benefit from the treatment of recombinant human EPO(53) . Recently, our data indicated that androgens can repress the expression of the TR2 orphan receptor mRNA in human prostate LNCaP cells and rat ventral prostate(4, 22) . It is possible that one of the potential androgenic effects for the induction of EPO expression may be indirectly involved in the suppression of the TR2 orphan receptor-mediated repression mechanism for EPO expression. Other possibilities include androgens directly increasing the EPO gene expression via the potential androgen response elements located at the 5`- or 3`-flanking region of the human EPO gene or stimulating the proliferation of erythrocytic progenitors in bone marrow(54, 55, 56) .
For several years, it was widely assumed that members of the steroid receptor superfamily were capable of binding to response DNA elements in three fundamentally different ways, monomeric, homodimeric, and heterodimeric categories(57) . A monomeric receptor can bind to a single copy of a core recognition motif (such as NGFI-B); two receptors can bind to two copies of a core consensus sequence, resulting in either homo- or heterodimers (such as glucocorticoid receptor and RAR-RXR, respectively). We initially tried to investigate whether the homodimerization occurs between the EPO 3`-enhancer and the TR2 orphan receptor. Our results, however, indicate that the TR2 orphan receptor and its different deletion variants may individually recognize the EPO enhancer element (Fig. 4). Furthermore, we were unable to detect any potential interaction between the full-length TR2 orphan receptor, RXR, and RAR using a direct repeat of the promoter of the cellular retinol-binding protein type II gene (58) as a probe(23) . This again ruled out the possibility that the TR2 orphan receptor could form heterodimers with either RXR or RAR. Thus far, there is no sufficient evidence to show the TR2 orphan receptor forms either homo- or heterodimers by itself or with other receptors. Moreover, we observed similar phenomena for that of the TR4 orphan receptor, a close relative of the TR2 orphan receptor, during the study of gene expression on the SV40 major late promoter(43) .
Our data also suggest that suppression of EPO gene expression by the TR2 orphan receptor is accomplished in a DNA-dependent manner. It is noted that the HREs for steroid receptors are structurally related but functionally different(59) . Based on the zinc finger model, five amino acid residues at the C-terminal region of the first zinc finger are designated as the P box which is important in protein-DNA interaction(59) . The TR2 orphan receptor contains Glu-Gly-Cys-Lys-Gly amino acid sequences in the P box, whereas the human androgen receptor belonging to a different subfamily of the HREs consists of Gly-Ser-Cys-Lys-Val amino acid sequences in the same box. Our data showed that the TR2 orphan receptor may repress the EPO gene expression via the 3`-enhancer region (Fig. 5). In contrast, our data also showed such repression could be abolished when the chimeric TR2/ARp/TR2 orphan receptor, a construct replacing only the P box of the DNA-binding domain in the TR2 orphan receptor with that of the androgen receptor, was tested. These results highlight the importance and specificity of the protein-DNA interaction during EPO regulation by the TR2 orphan receptor.
In addition to several hypoxia-inducible nuclear factors discovered thus far, a few orphan receptors have been reported to be involved in the key regulation of EPO gene expression via the 3`-enhancer(35, 39) . Based on our data described above, we hypothesize that the repression model of the TR2 orphan receptor in EPO gene transcription is similar to that of COUP family members(39) . Thus, the TR2 orphan receptor may be one of the contributors, among the complicated network for binding the same DNA sequence of the EPO gene, and specifically compete with activators, such as transcriptional factors or orphan receptors (e.g. hepatic nuclear factor 4). The degree of inhibition could be dependent on which factor is in excess during hypoxia. As yet, we have no evidence that the TR2 orphan receptor can antagonize any positive regulator by competing for binding to the EPO enhancer. Therefore, the relative levels of the TR2 orphan receptor and other activators may control the switch of EPO production in response to hypoxia or cobalt.
It has long been known that EPO gene expression is highly tissue-specific and its expression levels increase in response to the severity of anemia(28, 29, 30) . By using in situ hybridization, Koury et al.(29) showed that EPO mRNA positive cells were not seen in any glomeruli, but were easily seen in tubular areas within the basement membrane in the anemic kidney. However, nonanemic kidneys even in the same section on the same slide exhibited no such labeling in either the cortex or medulla(29) , indicating that EPO expression is very low under normal conditions and may be undetectable by some methodologies including in situ hybridization. In addition, cell types expressing EPO in the kidney and liver assayed by in situ hybridization have been well documented(29) , and we, therefore, believe it is appropriate to compare directly the expression pattern of the TR2 orphan receptor with that of EPO as revealed by Koury's group.
Our in situ hybridization
results showed that both the TR2 orphan receptor and EPO transcripts
could be co-localized in the liver, kidney, and brain with the
expression pattern correlated with the liver-to-kidney shift of EPO
production during mouse development. In addition, the earliest TR2
orphan receptor expression in the mouse embryo was detected at 9.5
dpc, while the ontogeny for EPO is not exactly defined. In
transgenic mice with the EPO-null mutation, embryos died at about 13
dpc(60) , suggesting that the timing for both EPO and the TR2
orphan receptor expression may be correlated. However, the tissue
distribution of the TR2 orphan receptor in the mouse is much broader
than that of EPO and is proposed to be position-specific, instead of
tissue-specific.
Thus, the TR2 orphan receptor transcripts
are highly expressed in most tissues undergoing early but not terminal
differentiation.
One possible explanation is that the TR2
orphan receptor may have several target genes with EPO being just one
of them. The physiological significance of interaction between the TR2
orphan receptor and EPO can be revealed by the overexpression of the
TR2 orphan receptor in transgenic mice to see whether these transgenic
mice develop an anemic phenotype.
In summary, our data indicate that the EPO gene is the first identified human target gene regulated by the TR2 orphan receptor. In addition, the TR2 orphan receptor may function as a repressor in the complicated EPO gene regulation. The finding and characterization of the TR2RE-EPO here may further help us to isolate more physiological target genes of the TR2 orphan receptor in the future.