Win-Jing Young

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

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 RARβ 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.

Differential regulation of direct repeat 3 vitamin D3 and direct repeat 4 thyroid hormone signaling pathways by the human TR4 orphan receptor.

In situ hybridization analysis demonstrated that abundant testicular orphan receptor (TR4) transcripts were detected in kidney, intestine, and bone, which are vitamin D3 target organs. Cell transfection studies also demonstrated that the expression of the vitamin D3 target gene, 25-hydroxyvitamin D3 24-hydroxylase, can be repressed by TR4 through high affinity binding (K d = 1.32 nm) to the direct repeat 3 vitamin D3 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 (DR4T3RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T3RE showed a distinct supershifted pattern, and proteolytic analysis further demonstrated distinct digested peptides with either TR4-DR3VDRE or TR4-DR4T3RE. These results may therefore suggest that TR4 can adapt to different conformations once bound to DR3VDRE or DR4T3RE. The consequence of these different conformations of TR4-DR3VDRE and TR4-DR4T3RE may allow each of them to recruit different coregulators. The differential repression of TR4-mediated DR3VDRE and DR4T3RE 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.

Induction of the intronic enhancer of the human ciliary neurotrophic factor receptor (CNTFRalpha) gene by the TR4 orphan receptor. A member of steroid receptor superfamily

A conserved hormone response element, CNTFR-DR1 (5′-GGG-3′), has been identified in the 5th intron of the α component of the ciliary neurotrophic factor receptor (CNTFRα) gene for the human TR4 orphan receptor (TR4). Electrophoretic mobility shift assay showed a specific binding with high affinity (Kd = 0.066 nM) between TR4 and the CNTFR-DR1. A reporter gene assay using chloramphenicol acetyltransferase demonstrated that the 5th intron of CNTFRα has an enhancer activity which could be induced by TR4 in a dose-dependent manner. Furthermore, our in situ hybridization data showed that abundant TR4 transcripts were detected in adult brain, in regions of cortical and hippocampal neurons, as well as in many developing neural structures, including brain, spinal cord, ganglia (sympathetic and sensory), and neuronal epithelia (retinal, otic, olfactory, and gustatory). The striking similarities in the expression patterns of TR4 and CNTFRα in the developing and postnatal nervous systems further support the potential role of TR4 in neurogenesis. Collectively, these data suggest that the human CNTFRα gene could represent the first identified neural-specific gene induced by TR4.

A Bidirectional Regulation between the TR2/TR4 Orphan Receptors (TR2/TR4) and the Ciliary Neurotrophic Factor (CNTF) Signaling Pathway*

Previously, we reported that the nuclear orphan receptor TR4 could induce transcriptional activity via the 5th intron of the ciliary neurotrophic factor (CNTF) α receptor gene (CNTFR-I5). Here we show CNTF could increase TR4 expression and enhance the DNA-binding capacity of TR4. Interestingly, the expression of TR2, a close family member of TR4, could also be induced by CNTF. In return, TR2 induced CNTFRα transcriptional activity through binding to a direct repeat response element of AGGTCA within CNTFR-I5. The possibility of this mutual influence between TR2 and the CNTF signaling was further strengthened by in situ hybridization. Similar expression patterns of TR2 and CNTFRα were observed in most of the developing neural structures such as the ganglia, neural epithelia, spinal cord, and the periventricular areas of brain. Together, our data suggest that an interaction between TR2/TR4 and the CNTF signaling pathway may occur, supporting the hypothesis that TR2/TR4 may play important roles in neurogenesis.

Ontogeny and autoregulation of androgen receptor mRNA expression in the nervous system

Androgens and the androgen receptor (AR) both play critical roles for the development of the male phenotype. To investigate the roles of androgens in the developing nervous system, we examined the AR messenger RNA distribution by in situ hybridization. Our results indicate that AR transcripts were detectable in male mouse embryos at embryonic day 11 (E11). Intensive AR labeling appears in the neuroepithelium of brain vesicles and spinal cord, as well as in the reproductive organs. During E15–E16, new and strong AR labeling appeared in the cortex of cerebrum and hippocampus. Specific AR signals were also present in the brain areas known for hormonal control of copulatory behavior and mediating sensory processing. Interestingly, many ganglia were found to express AR mRNA at E15–E16. These novel AR-expressing sites include the dorsal root, sympathetic, and celiac ganglia, as well as the opthalmic nerve of trigeminal ganglion. Sex dimorphism of AR expression in brain was also observed during E15–E16. Postnatally, brain and spinal cord can respond to circulating androgen levels by modifying their AR gene expression, but the ganglia cannot. Together, these data suggest androgens may have a great influence on the development and maintenance of the nervous system through the AR.

Induction of an intronic enhancer of the human ciliary neurotrophic factor receptor (CNTFRα) gene by the TR3 orphan receptor

A hormone response element, CNTFRα-NBRE (5′-AAAGGTCA-3′) has been identified in the fifth intron of the α component of ciliary neurotrophic factor receptor gene (CNTFR-I5) for the human TR3 orphan receptor (TR3). A specific binding between in vitro expressed TR3 and CNTFRα-NBRE was demonstrated by using electrophoretic mobility shift assay. A reporter gene assay using chloramphenicol acetyltransferase (CAT) showed that CNTFR-I5 has an enhancer activity that could be induced by TR3 in a dose-dependent manner. This induction was significantly reduced in the absence of CNTFRα-NBRE. Together, these results indicate CNTFRα-NBRE is sufficient to mediate TR3 action in inducing the enhancer activity of CNTFR-I5. Our finding may, therefore, suggest CNTFRα is a target gene regulated by TR3 and expand the role of TR3 in the nervous system.

Isolation and characterization of the androgen receptor mutants with divergent transcriptional activity in response to hydroxyflutamide

A yeast genetic screening was developed to isolate androgen receptor (AR) mutants with divergent transactivation characteristics in response to hydroxyflutamide (HF), an active metabolite of flutamide used for prostate cancer treatment. Two mutants carrying the substitution C685Y or E708K were isolated and characterized. Substitution of C685Y for wild-type AR (wtAR) rendered the receptor supersensitive to androgenic activity from HF and female hormones such as 17β-estradiol (E2) and progesterone (P). Similar effects were observed in the AR mutant, named T876AAR, isolated from LNCaP cells. Surprisingly, we found that C685YAR7, but not T876AAR7, could be activated by casodex (bicalutamide), a nonsteroidal pure antiandrogen, with an induction fold 3- to 5-fold times higher than that for wild type or T876AAR. By contrast, although replacement of E708K for wtAR showed little effect on dihydrotestosterone-mediated transactivation, E708KAR lost its transcriptional response from many other ligands. The effects of ligands on E708KAR could be controlled at the DNA-binding level owing to the finding of a significant decrease in the DNA-binding ability once E708KAR was bound to HF, E2, or P. Together, these results suggest that C685YAR can be a novel tool for assaying the androgenic activity from antiandrogens, and the mechanism revealed from E708KAR could provide a possible explanation for the partial androgen insensitivity syndrome in men with a natural E708KAR mutation.

Androgen effects on the solubility and conformational change of the androgen receptor in baculovirus expression system

To purify the androgen receptor (AR) efficiently from baculovirus expression system, we fused 6 histidine residues with the N-terminal domain of AR as a tag to specifically bind to Ni+2-affinity column. Our data indicated that adding androgen can increase the binding capacity of his-tag AR to the Ni+2-affinity column, and this increased binding capacity of AR could be due to the exposure of histidine residues of N-terminal domain induced by androgen. The androgen-enhanced binding to Ni+2-column also correlated with the increasing solubility of AR. Electrophoretic mobility shift assay further indicated that only purified AR could interact with androgen response element. Together, our data suggest that the binding of androgen to the hormone binding domain of AR may result in the conformational change of the N-terminal domain of AR and increase the hydrophilic property of AR.

Monoclonal anti-androgen receptor antibodies: production, characterization and potential diagnostic applications.

Several monoclonal antibodies (mAbs) and novel mAb-based assays for the androgen receptors (AR) have been developed. Large amounts of the recombinant human AR protein produced by a baculovirus expression system were used as an antigen to produce mAbs. Twenty-nine AR-specific mAbs were first confirmed by Western blot analysis and were then characterized for their immunoglobulin isotypes, epitopes, and epitope localization in AR. Novel assays using flow cytometry and sandwich enzyme-linked immunosorbent assays (ELISA) were established to detect AR-expressing cells and to quantify soluble AR protein, respectively. Using immunostaining, we identified several anti-AR mAbs exclusively recognizing AR within the nuclei of the prostate cancer cell line LNCaP and of prostate tissues in both frozen and paraffin-embedded sections, whereas other mAbs could detect AR in both nuclear and cytoplasmic compartments. Interestingly, certain mAbs, such as G122-25 and G122-77, could distinguish the androgen-bound AR from the unoccupied AR. In sum, many purified AR protein and anti-AR mAbs, together with the assays developed, could be powerful tools for the study of functional AR and for the diagnosis of prostatic cancers.