Negative Feedback Control of the Retinoid-Retinoic Acid/Retinoid X Receptor Pathway by the Human TR4 Orphan Receptor, a Member of the Steroid Receptor Superfamily

Abstract

Amino acid sequence analysis indicates that the human TR4 orphan receptor (TR4) is a member of the estrogen/thyroid receptor subfamily of the steroid/thyroid receptor superfamily and recognizes the AGGTCA direct repeat (DR) of the hormone response element. Here we demonstrate using the electrophoretic mobility shift assay that TR4 binds specifically to DR with a spacing of 1 and 5 base pairs (DR1 and DR5), which are the response elements for retinoic acid receptor (RAR) and retinoid X receptor (RXR), respectively. A reporter gene assay using chloramphenicol acetyltransferase demonstrated that TR4 repressed RA-induced transactivation in a TR4 dose-dependent manner. Inhibition of the retinoid signal pathway also occurs through natural response elements found in CRBPII and RAR␤ genes. Our data suggest that the mechanism of repression may not involve the formation of functionally inactive heterodimers between TR4 and RAR or RXR. Instead, we show that TR4 may compete for hormone response elements with RAR and RXR due to its higher binding affinity. Furthermore, treatment of F9 murine teratocarcinoma (F9) cells with 10 ؊6 M all-trans-retinoic acid increased TR4 mRNA levels, and this change was accompanied by an increased amount of endogenous TR4 protein that can bind to RXRE in electrophoretic mobility shift assay. Our data therefore strongly suggest that the retinoid signal pathway can be regulated by TR4 in a negative feedback control mechanism, which may restrict retinoic acid signaling to certain elements in a cell-specific fashion.The steroid/thyroid hormone receptor superfamily is a large group of related transcriptional factors that control cellular differentiation, development, and homeostasis by direct interaction with distinct cis-elements in target genes (1). This superfamily includes receptors for steroids, thyroid, vitamin D 3 , retinoids, and a large number of orphan receptors whose cognate ligands are still unknown (2). Regardless of whether transcriptional activity is controlled by ligand binding, each of these proteins is able to bind to specific DNA sequences called hormone response elements (HREs) 1 in their target genes. The sequence-specific DNA binding properties of nuclear receptors are determined by their highly conserved DNA binding domains (DBD). The P box of the DBD formed by three amino acids at the C-terminal base of the first zinc finger is responsible for the recognition of response elements. Based on the sequence within the P box and the generic recognition sequence of the P box for the HRE, the steroid/thyroid hormone receptor superfamily can be divided into the GR and ER/TR subfamilies (3). The GR group, which includes GR, MR, PR, and AR, prefers to bind to the glucocorticoid response element (5Ј-AGAACAnnnTGTTCT-3Ј) palindromic consensus sequence. Other receptors that have glutamic acid and glycine at the first two positions of the P box are assigned to the ER/TR subfamily. This subfamily, which includes ER, T 3 R, VD 3 R, RARs, RXRs, and most of the orphan receptors, recognize the ...

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation

“…In vitro data suggest that TR4 functions as a master regulator to modulate many signaling pathways, including induction of the ciliary neurotrophic factor alpha [5, 6], interfering with retinoic acid receptor/retinoid X receptor [7], thyroid receptor [8], androgen receptor [9], and estrogen receptor-mediated pathways [10], and facilitating viral infection and propagation of HPV-16 and SV40 [11]. Mice lacking Tr4 ( TR4KO ) have high rates of early postnatal mortality, show significant growth retardation [12], display reproductive defects in both genders [12, 13], abnormalities in spermatogenesis [14], reduced lipoprotein metabolism [15, 16], and reduced gluconeogenesis [17], as well as defects in cerebella development [18].…”

Suppression of Hepatitis B Virus Core Promoter by the Nuclear Orphan Receptor TR4

“…Our previous data also showed that the TR4 has a better affinity than RXR␣ to recognize the DR1-HRE in the promoter of the cellular retinol-binding protein II gene (21). To examine whether TR4 can bind to the potential DR1-HRE (named TR4RE-HBV, nucleotide coordinates 1751-1778 of the HBV ayw DNA sequence) on the HBV core promoter (13), the electrophoretic mobility shift assay (EMSA) was performed with in vitro translated TR4 protein, using radiolabeled TR4RE-HBV oligonucleotides as probes.…”

“…These findings suggest that these nuclear receptors in the liver can coordinately regulate gene expression by interacting with HRE sequences in the promoters of their target genes. Our data show that TR4 can interfere with a number of other nuclear receptors by competing for binding to the same HREs (21,22).…”

“…For example, TR4 induces reporters containing the DR1 site of human ciliary neurotrophic factor ␣ receptor promoter and the DR4 site of the ␣-myosin heavy chain promoter (19,20). In contrast, TR4 suppresses RXR␣/retinoic acid receptor (DR1), vitamin D3 receptor (DR3), and RXR␣/PPAR␣ (DR1)-mediated transactivation as shown by the reporter assay (21)(22)(23). These results suggest that TR4 may act as a negative regulator of its target genes containing hormone response elements (HREs) by competition with other nuclear receptors for binding to the HREs (21,23).…”

“…In contrast, TR4 suppresses RXR␣/retinoic acid receptor (DR1), vitamin D3 receptor (DR3), and RXR␣/PPAR␣ (DR1)-mediated transactivation as shown by the reporter assay (21)(22)(23). These results suggest that TR4 may act as a negative regulator of its target genes containing hormone response elements (HREs) by competition with other nuclear receptors for binding to the HREs (21,23). In addition, our previous data showed that TR4 could also suppress androgen receptor-mediated transactivation via protein-protein interaction (22).…”