Suppression of the Human Erythropoietin Gene Expression by the TR2 Orphan Receptor, a Member of the Steroid Receptor Superfamily

Abstract

A DNA response element, TR2RE-EPO (5-TCTGAC-CTCTCGACCTAC-3) has been identified in the 3-minimal hypoxia-inducible enhancer of the human erythropoietin gene for the TR2 orphan receptor, an androgenrepressed transcription factor and a member of the steroid/thyroid hormone receptor superfamily. Electrophoretic mobility shift assay showed a specific binding with high affinity (K d ‫؍‬ 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 3 , 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 Refs. 6 and 7). Thus, they share common modular architecture within the superfamily, including a variable Nterminal 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-⌬ 12,14 -prostaglandin J 2 , and melatonin (5-methyoxy-N-acetyltryptamine) for retinoid X receptor (RXR), 1 peroxisome proliferator-activated receptor ␥, and retinoid Z receptors ␣ and ␤ (8 -11). In addition, some orphan receptors are constitutive transactivators or repressors, such as the TR3 orphan receptor or COUP-TF I (12-17). 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 pathwa...

The effects of iron deficiency on estradiol-induced suppression of erythropoietin induction in rats: implications of pregnancy-related anemia

“…54 Other than that, some previous reports suggest that the effect of E 2 on Epo gene regulation acts through steroid hormone receptors; there are tandem repeats of steroid hormone receptor response element half-sites in the Epo promoter and enhancer regions, 55 and the testicular receptor 2 (TR2) orphan receptor, which shares structural homology with members of the steroid/ thyroid hormone receptor superfamily, suppresses Epo gene expression. 56 Another recent report demonstrated that E 2 attenuated Epo expression by interfering with hypoxic increases in HIF-1␣ protein through an estrogen receptor-dependent mechanism. 57 However, the results indicating possible involvement of steroid hormone receptors or HIF-1␣ were all obtained from in vitro experiments, and we actually observed that E 2 did not inhibit Epo induction in vitro using Epo-producing Hep3B cells derived from human hepatoma cells (data not shown).…”

Alterations of NFIA in chronic malignant myeloid diseases

“…Deletion in a PV is coherent with the known function of NFIA in erythropoiesis. [7][8][9] The calculated number of NFIA gene copy suggests that the deletion was present at the heterozygous state in almost all cells or homozygous in around half the cells. The moderate JAK2 V617F allele burden suggests that the mutation may have occurred on the background of the 1p31.3 deletion.…”

“…5,6 NFIA regulates erythropoiesis through a network involving NR2C1, GATA1 and erythropoietin. [7][8][9] To determine whether NFIA could have a function in early leukemogenesis, we searched for alterations of the NFIA gene in 89 chronic hematopoietic diseases by using array-comparative genomic hybridization (aCGH) and DNA sequencing. Our series comprised 35 chronic myelomonocytic leukemias and 54 myeloproliferative disorders including 8 cases of polycythemia vera (PV), 42 cases of essential thrombocythemia and 4 cases of idiopathic myelofibrosis.…”

Cloning and Characterization of Mouse RIP140, a Corepressor for Nuclear Orphan Receptor TR2

“…It is also known that this gene encodes two isoform receptors, one retaining the entire ligand binding domain (LBD) and the other truncated at the LBD, which exhibit differential expression patterns in developing testes (24). The biological function of the full-length TR2 has been examined in a variety of systems, such as the cellular RA binding protein I promoter containing a direct repeat 4 (DR4) element (6), a RAR-responsive element (RARE) of the DR5 type from RAR␤ (24, 30), a DR2 from the simian virus 40 (SV40) promoter (26), and a DR2 from the erythropoietin gene promoter (27). In all tested systems, the full-length TR2 consistently represses reporter gene expression in cell cultures supplemented with either regular serum or charcoal-depleted serum.…”