Differential Regulation of Direct Repeat 3 Vitamin D3and Direct Repeat 4 Thyroid Hormone Signaling Pathways by the Human TR4 Orphan Receptor

Abstract

In situ hybridization analysis demonstrated that abundant testicular orphan receptor (TR4) transcripts were detected in kidney, intestine, and bone, which are vitamin D 3 target organs. Cell transfection studies also demonstrated that the expression of the vitamin D 3 target gene, 25-hydroxyvitamin D 3 24-hydroxylase, can be repressed by TR4 through high affinity binding (K d ‫؍‬ 1.32 nM) to the direct repeat 3 vitamin D 3 receptor response element (DR3VDRE). This TR4-mediated repression of DR3VDRE is in contrast to our earlier report that TR4 could induce thyroid hormone target genes containing a direct repeat 4 thyroid hormone response element (DR4T 3 RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T 3 RE showed a distinct supershifted pattern, and proteolytic analysis further demonstrated distinct digested peptides with either TR4-DR3VDRE or TR4-DR4T 3 RE. These results may therefore suggest that TR4 can adapt to different conformations once bound to DR3VDRE or DR4T 3 RE. The consequence of these different conformations of TR4-DR3VDRE and TR4-DR4T 3 RE may allow each of them to recruit different coregulators. The differential repression of TR4-mediated DR3VDRE and DR4T 3 RE transactivation by the receptor interacting protein 140, a TR4 coregulator, further strengthens our hypothesis that the specificity of gene regulation by TR4 can be modulated by protein-DNA and protein-protein interactions.Steroid hormones are key physiological mediators of development and homeostasis (1-4). Understanding the crosstalk between steroid hormone-dependent and -independent signaling pathways is critical for gaining further insight into the integration of cellular regulatory cues that modulate development and tissue-specific gene expression. The biological effects of steroids and related hormones, including derivatives of vitamins A and D 3 , are mediated through their cognate receptors (1-4). These receptors are members of a large group of ligandactivated proteins that act as transcriptional activators or repressors. However, there is another group of nuclear receptors that shares the same molecular structure as steroid hormone receptors but has no known ligands. The members of this group have therefore been named orphan receptors (5). A common characteristic of many of these orphan receptors is that they exert at least part of their function as regulators of other receptors. This may occur by several different mechanisms: competition for the same response element, heterodimer formation with the regulated receptor, or heterodimer formation with the retinoid X receptor (RXR), 1 thus titrating out available RXR protein. For instance, chicken ovalbumin upstream promoter transcription factors have been demonstrated to suppress ligand-induced gene activation, including that of vitamin A, vitamin D 3 , and thyroid hormone target genes (6). This interfering effect might involve the formation of a transcriptional silencing complex with RXR or competition...

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

“…TR4 is a transcriptional factor that recognizes the DNA element composed of the direct repeat (DR) motif located regulatory regions of its target genes, including DR-3 [8]. As shown in Fig.…”

“…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].…”

TR4 Inhibits LXR-mediated Decrease of Lipid Accumulation in 3T3-L1 Adipocytes

“…A common characteristic of various nuclear receptors is that they play roles as functional regulators of other nuclear receptors since several nuclear receptors use a common response element for the regulation of target gene expression (Lee et al, 1997; Lee et al, 1999). These suggest that mutual regulation between nuclear receptors in the intracellular environment of adipocytes may lead to control adipocyte development and lipid homeostasis.…”

“…Testicular orphan nuclear receptor 4 (TR4; Nr2c2), a member of the nuclear receptor superfamily, is able to regulate the expression of target genes through binding to direct repeats (DRs) of AGGTCA core motifs with variable numbers of spacer nucleotides (Kim et Lee et al, 1999). Several studies suggested that TR4 may play key roles in glucose and lipid metabolism (Liu et al, 2007).…”

Conjugated Linoleic Acid Negatively Regulates TR4 Activity in 3T3-L1 Adipocytes

“…Perilipin, a lipid-droplet associated protein, coats lipid droplets in differentiating adipocytes and restricts the access of cytosolic lipases to lipid droplets, resulting in promotion of triacylglycerol storage (Brasaemle et al, 2000). Early studies have shown that TR4 could modulate gene expression via binding to DR1 element although TR4 has broad affinity for variable DRs with different spacer sequences from 0 to 6 nucleotides (DR0-DR6) (Lee et al, 1995; Lee et al, 1997; Lee et al, 1999). Here, we show that TR4 regulates perilipin gene expression through DR1 element (Plin-TR4RE) located in the perilipin promoter.…”