Novel nonsecosteroidal vitamin D receptor modulator inhibits the growth of LNCaP xenograft tumors in athymic mice without increased serum calcium.

BACKGROUND: We recently reported on novel vitamin D receptor (VDR) modulators that are structurally distinct from the secosteroid 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the endogenous activator of VDR. One of these compounds, LG190119, was tested for the ability to inhibit the growth of LNCaP human prostate cancer cell-derived tumors in athymic mice. METHODS: In one study, athymic mice with established LNCaP xenograft tumors were dosed orally every day with LG190119 (3 or 10 mg/kg) or with a synthetic analog of 1,25(OH)(2)D(3), EB1089 (1 microg/kg), for 15 days. In another study ("prevention mode"), oral administration (every other day) of 10 mg/kg LG190119 or a non-hypercalcemic dose of 1,25(OH)(2)D(3) (0.5 microg/kg) was initiated prior to tumor development and continued for 84 days. In both studies, tumor volumes, mouse weights, and serum calcium levels were measured. RESULTS: In the established tumor study, LG190119 at each dose resulted in significant tumor growth inhibition without hypercalcemia at both 10 and 15 days. EB1089 treatment resulted in significant tumor growth inhibition only at Day 10 and resulted in hypercalcemia at Day 15. In the prevention-mode study, LG190119 markedly slowed tumor growth without increased serum calcium in comparison with either vehicle or 1,25(OH)(2)D(3) treatment (P < 0.001). CONCLUSIONS: LG190119 effectively inhibited LNCaP xenograft tumor growth without increased serum calcium levels or any other apparent side effects. Compounds of this class may represent promising new therapeutics for treatment of prostate cancer and other cancers with fewer undesirable side effects than currently used drugs.

Coexpression of ER beta with ER alpha and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment.

Estrogen and progestin receptors (ER, PgR) play a critical role in the regulation of neuroendocrine functions in females. The neuroanatomical distribution of the recently cloned, ER beta, overlaps with both ER alpha and PgR. To determine whether ER beta is found within ER alpha- or PgR-containing neurons in female rat, we used dual label immunocytochemistry. ER beta-immunoreactivity (ER beta-ir) was primarily detected in the nuclei of cells in the periventricular preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr), the paraventricular nucleus, the supraoptic nucleus, and the medial amygdala (MEApd). Coexpression of ER beta-ir with ER alpha-ir or PgR-ir was observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2 treatment decreased the number of ER beta-ir cells in the PvPO and BNSTpr and the number of ER alpha-ir cells in the MEApd and paraventricular nucleus, and therefore decreased the number of cells coexpressing ER beta-ir and ER alpha-ir in the PvPO, BNSTpr, and MEApd. E2 treatment increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd, a portion of which also contained ER beta. These results demonstrate that ER beta is expressed in ER alpha- or PgR-containing cells, and they suggest that E can modulate the ratios of these steroid receptors in a brain region-specific manner.

The vitamin D response element-binding protein. A novel dominant-negative regulator of vitamin D-directed transactivation.

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a non-vitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin d-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamin d-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominant-negative regulator of vitamin D action.

Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1.

We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ERalpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERalpha chimera (GFP-ERalpha), we find that 17beta-estradiol (E2) changes the normally diffuse nucleoplasmic pattern of GFP-ERalpha to a hyperspeckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERalpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E2, 4-hydroxytamoxifen, or ICI 182,780 causes ERalpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E2 on the redistribution and solubility of GFP-ERalpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERalpha movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160). Using E2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERalpha pool. Experiments performed with bioluminescent derivatives of ERalpha and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERalpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.

Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor alpha403 heterodimers is developmentally regulated during myeloid differentiation.

The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARalpha exhibiting dominant-negative activity (RARalpha403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 microM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARalpha in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARalpha403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARalpha hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARalpha403 heterodimers that we observed at these different stages of myeloid development.

Estrogen receptor beta activates the human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen.

Human estrogen receptor-alpha (hERalpha) or -beta (hERbeta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERalpha and hERbeta, although raloxifene was more potent through ERalpha than ERbeta, and tamoxifen was more potent via ERbeta than ERalpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRARalpha-1) promoter through nonclassical EREs by a mechanism that was ERalpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERalpha, hERbeta did not induce reporter activity driven by the hRARalpha-1 promoter in the presence of estrogen. While hERbeta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERbeta was completely blocked by estrogen. Like ERalpha, transcriptional activation of this promoter by ERbeta was not mediated by direct receptor binding to DNA. While hERalpha was shown to act through two estrogen-responsive sequences within the promoter, hERbeta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERbeta via the hRARalpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERbeta from the hRARalpha-1 promoter in Hep G2 cells required the amino-terminal region of ERbeta, a region that was not necessary for estrogen-induced ERbeta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 microM) antagonist via hERalpha and as a fairly potent (IC50 approximately 200 nM) antagonist via hERbeta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERalpha or ERbeta in vitro, it did bind to ERbeta in whole-cell binding assays, and therefore, it is likely metabolized to an ERbeta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERbeta to stimulate the hRARalpha-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.

High-mobility group chromatin proteins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA binding in vitro and transcriptional activity in mammalian cells.

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors.

Retinoid X receptor (RXR) ligands activate the human 25-hydroxyvitamin D3-24-hydroxylase promoter via RXR heterodimer binding to two vitamin D-responsive elements and elicit additive effects with 1,25-dihydroxyvitamin D3.

We have previously shown that RNA levels of kidney 25-hydroxyvitamin D3-24-hydroxylase (24(OH)ase), a key metabolic enzyme for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is up-regulated by retinoids in mice within hours. Deletion analysis of approximately 5500 base pairs of the human 24(OH)ase promoter showed that the sequence between -316 and -142 contained the information necessary and sufficient for retinoid-induced activation of the promoter. This region contains two previously defined vitamin D-responsive elements (VDREs) at -294 to -274 and -174 to -151. Mutation of either VDRE diminished responsiveness of the -316 to -22 promoter sequence to retinoids or 1,25(OH)2D3, while mutation of both VDREs essentially abolished the activity of the ligands via the promoter. Heterologous promoter vectors driven by the VDREs were responsive to a retinoid X receptor (RXR)-selective ligand (LG100268), a retinoic acid receptor (RAR)-selective ligand (TTNPB), or 1,25(OH)2D3, while combinations of LG100268 with either TTNPB or 1,25(OH)2D3 resulted in additive increases in activity. Band shift analyses showed that vitamin D receptor, RAR, or RXR alone did not bind to the VDREs; however, the combination of either vitamin D receptor or RAR with RXR led to retardation of each of the labeled probes. Treatment of nontransfected CV-1 cells with retinoids or 1,25(OH)2D3 resulted in induction of 24(OH)ase RNA, and ligand combinations led to increased RNA levels. These data imply that either or both of the heterodimer partners can be occupied with ligand to induce this enzyme, with dual receptor occupation leading to increased activation.

Retinoic acid receptor alpha expression correlates with retinoid-induced growth inhibition of human breast cancer cells regardless of estrogen receptor status.

Retinoic acid receptor (RAR) alpha has been shown to play a role in retinoid-induced growth inhibition of human breast cancer cell lines that express the estrogen receptor (ER). The dogma in the field has been that ER-positive breast cancer cell lines respond to retinoid treatment because they express RAR alpha, whereas ER-negative breast cancer cell lines are refractory to retinoid treatment and have been thought to express little or no RAR alpha. We set out to test several ER-negative breast cancer cell lines for expression of RAR alpha protein and responsiveness to retinoids in growth inhibition assays. Of six ER-negative breast cancer cell lines that were tested, one (SK-BR-3) had high levels of RAR alpha protein as measured by ligand-binding immunoprecipitation (approximately 55 fmol/mg protein) and also displayed sensitivity to growth inhibition by retinoids (9-cis-retinoic acid; EC50, approximately 3 nM). These cells were more sensitive than an ER-positive cell line, T-47D, which expressed approximately 35 fmol RAR alpha/mg total protein (9-cis retinoic acid; EC50, approximately 50-100 nM). Another ER-negative cell line, Hs578T, also expressed RAR alpha (approximately 23 fmol/mg) and was sensitive to retinoid-induced growth inhibition, albeit to a lesser extent than SK-BR-3 or T-47D cells. In contrast, the other ER-negative cell lines tested expressed low (<10 fmol/mg) or no detectable levels of RAR alpha protein and also did not respond to retinoids in growth inhibition assays. A RAR alpha agonist displayed 100 times greater potency than a RARgamma agonist in growth inhibition of both T-47D and SK-BR-3 cells, suggesting RAR alpha involvement in the process. Furthermore, a RAR alpha antagonist completely abolished the growth inhibition induced by RAR agonists, implying that the activity of the agonists is exerted solely through RAR alpha, not RARgamma, which is also expressed in both cell lines. Additionally, although retinoid X receptor (RXR) compounds are weakly active in growth inhibition of the RAR alpha-positive cell lines, they markedly increased the growth-inhibitory activity of RAR ligands. RXR compounds also potentiated the action of the antiestrogen 4-hydroxytamoxifen to inhibit the growth of T-47D cells. These findings have clinical ramifications in that patients with ER-negative tumors that are RAR alpha positive may be candidates for retinoid therapy. Additionally, combinations of RXR ligands with RAR ligands (especially RAR alpha agonists) and/or antiestrogens may have utility in the treatment of breast cancer.

1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1.

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.

Vitamin D and gonadal steroid-resistant New World primate cells express an intracellular protein which competes with the estrogen receptor for binding to the estrogen response element.

New World primates (NWP) exhibit a form of compensated resistance to vitamin D and other steroid hormones, including 17beta-estradiol. One postulated cause of resistance is that NWP cells overexpress one or more proteins which block hormone action by competing with hormone for its cognate hormone response element. Here we report that both nuclear and postnuclear extracts from NWP, but not Old World primate, cells contained a protein(s) capable of binding directly to the estrogen response element (ERE). This ERE binding protein(s) (ERE-BP) was dissociated from the ERE by excess of either unlabeled ERE or excess of the ERE half-site motif AGGTCAcag. DNA affinity chromatography using concatamers of the latter resulted in > 20,000-fold purification of the ERE-BP. The intensity of the ERE-BP-ERE complex in electromobility shift assay was indirectly related to the amount of wild-type Old World primate estrogen receptor (ER) but not affected when potential ligands, including 17beta-estradiol (up to 100 nM), or anti-ER antibody was added to the binding reaction. We conclude that vitamin D-resistant and gonadal steroid-resistant NWP cells contain a protein(s) that may "silence" ER action by interacting directly with the ERE and interfering with ER binding.

Elevated expression of retinoic acid receptor-alpha (RAR alpha) in estrogen-receptor-positive breast carcinomas as detected by immunohistochemistry.

Retinoids modulate gene activity, cell growth and differentiation by binding to a series of nuclear receptors, i.e., retinoic acid receptors (RARs) or retinoid X receptors. Retinoic acid (RA) inhibition of estrogen receptor (ER)-positive breast carcinoma seems to be mediated through RAR alpha. Estrogens upregulate RAR alpha in ER-positive breast carcinoma cell lines. In this study we examined RAR alpha expression in the ER-positive MCF7 and ER-negative MDA-MB-231 human breast carcinoma cell lines as well as in 10 ER-negative and 9 ER-positive infiltrating ductal breast carcinoma specimens using immunohistochemistry and quantitation by image cytometry. MCF7 cells expressed twofold higher levels of RAR alpha protein than MDA-MB-231 cells. RAR alpha expression, as detected by immunostaining and quantitated by image cytometry, was upregulated in these cells by estradiol. ER-positive breast carcinoma specimens also exhibited approximately two-fold higher RAR alpha levels than their ER-negative counterparts. Thus, RAR alpha expression is significantly elevated in ER-positive breast tumors as assessed by detection and quantitation using immunohistochemical staining and image cytometry, respectively. Whether the decrease in RAR alpha protein levels and loss of RA-mediated growth inhibition in ER-negative tumor plays a role in the increased metastatic potential of ER-negative tumors remains to be determined.

Estrogen and estrogen receptor antagonists stimulate transcription from the human retinoic acid receptor-alpha 1 promoter via a novel sequence.

We and others previously reported that up-regulation of retinoic acid receptor-alpha (RAR alpha) RNA and protein levels is elicited by estrogen in human breast cancer cells. We set out to determine the mechanism by which estrogen up-regulates RAR alpha. Cloning of 500 bp of the human (h) RAR alpha 1 promoter has been reported previously; we obtained this 500-bp DNA sequence by PCR techniques from human genomic DNA and tested its activity in the context of a luciferase-containing reporter vector in Hep G2 cell contransactivation assays. Estradiol elicited a 6- to 8-fold increase in luciferase activity from the reporter vector driven by hRAR alpha promoter sequence between -491 and +36 bp that was dependent on the presence of contransfected estrogen receptor (ER). Analysis of various truncated versions of this promoter sequence indicated that two regions of the sequence are sensitive to estrogen stimulation. The first resides in the region -49 to -79 bp upstream from the transcription start site and conferred approximately 2-fold activation by estrogen. This region does not contain a consensus estrogen response element, and ER binding to this DNA sequence was not observed. The second responsive sequence lies at -455 to -491 bp and conferred in additional 4- to 6-fold activation by estrogen. This upstream sequence contains two A/TGGTCA half-sites; however, direct binding of ER to this sequence was not observed. Additionally, ER DNA-binding domain mutants that are not capable of binding to DNA were just as effective as wild type ER in their ability to confer estrogen responsiveness to the RAR alpha promoter, implying that ER DNA-binding ability is not required for the estrogen-induced increase in transcriptional activity. Mutation of either half-site or of an additional immediate downstream sequence in the context of the -491 to +36 bp construct reduced the luciferase activity induction by estrogen from 6-fold to 1.5- to 2-fold. Placement of the region between -455 to -491 bp upstream of an SV40 promoter-driven luciferase vector conferred approximately 20- to 30-fold stimulation of luciferase activity by estrogen in an ER-dependent manner. The ER antagonists, 4-hydroxy-tamoxifen, keoxifene, and ICI 164384, each acted as weak agonist via the hRAR alpha promoter in contransactivation assays, exhibiting 20-30% of the efficacy that was demonstrated by estradiol. Interestingly, upon treatment of MCF7 cells with estradiol or the ER antagonists, increased levels of RAR alpha RNA and protein were observed with the antagonists as well as with estrogen.

Inhibition of vitamin D receptor-retinoid X receptor-vitamin D response element complex formation by nuclear extracts of vitamin D-resistant New World primate cells.

Most New World primate (NWP) genera evolved to require high circulating levels of steroid hormones and vitamin D. We hypothesized that an intracellular vitamin D binding protein (IDBP), present in both nuclear and cytoplasmic fractions of NWP cells, or another protein(s) may cause or contribute to the steroid hormone-resistant state in NWP by disruption of the receptor dimerization process and/or by interference of receptor complex binding to the consensus response elements present in the enhancer regions of steroid-responsive genes. We employed electromobility shift assay (EMSA) to screen for the presence of proteins capable of binding to the vitamin D response element (VDRE). Nuclear and post-nuclear extracts were prepared from two B-lymphoblastoid cell lines known to be representative of the vitamin D-resistant and wild type phenotypes, respectively. The extracts were compared for their ability to retard the migration of radiolabeled double stranded oligomers representative of the VDREs of the human osteocalcin and the mouse osteopontin gene promoters. A specific, retarded band containing VDR-RXR was identified when wild type cell but not when vitamin D-resistant cell nuclear extract was used in the binding reaction with either probe. In addition, vitamin D-resistant cell nuclear extract contained a protein(s) which was bound specifically to the VDRE and was capable of completely inhibiting VDR-RXR-VDRE complex formation; these effects were not demonstrated with nuclear extract from the wild type cell line or with the post-nuclear extract of the vitamin D-resistant cell line. We conclude that a VDRE-binding protein(s), distinct from IDBP and present in nuclear extract of cells from a prototypical vitamin D-resistant NWP, is capable of inhibiting normal VDR-RXR heterodimer binding to the VDRE.

Retinoid X receptor acts as a hormone receptor in vivo to induce a key metabolic enzyme for 1,25-dihydroxyvitamin D3.

We demonstrate here that RNA levels of 25-hydroxy-vitamin D3-24-hydroxylase (24-(OH)ase), a key catabolic enzyme for 1,25-dihydroxyvitamin D3, are increased by a highly selective retinoid X receptor (RXR) ligand, LG100268, in mice within hours. Correspondingly, upon LG100268 treatment, kidney 24-(OH)ase enzymatic activity increases 5-10-fold. The endogenous retinoid hormones, all-trans-retinoic acid and 9-cis-retinoic acid, and the synthetic retinoic acid receptor-selective compound, TTNPB, also stimulate 24-(OH)ase. Additionally, we show that LG100268 stimulates transcription of a luciferase reporter plasmid driven by 24-(OH)ase promoter sequences in the presence of RXR in CV-1 cell cotransactivation assays. This first demonstration of a gene that is regulated in the intact animal through an RXR-mediated pathway confirms earlier hypotheses that RXR is a bona fide hormone receptor. Regulation of a key gene in the vitamin D signaling pathway by a retinoid transducer may provide a molecular basis for some of the documented biological effects of vitamin A on bone and vitamin D metabolism.

Sensitive and specific detection of retinoid receptor subtype proteins in cultured cell and tumor extracts.

Subtype-specific antipeptide antibodies have been developed against each of the retinoic acid receptors (RARs alpha, beta, and gamma) and each of the retinoid X receptors (RXRs alpha, beta, and gamma). Each antibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by both immunoblot and immunoprecipitation technology. We describe a sensitive and specific assay that combines the binding of cultured cell and tumor extracts to [3H]all-trans-retinoic acid or [3H]9-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by the subtype-specific antibodies to determine the levels of functional retinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compounds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six retinoid receptor proteins ranging in concentration from 9 fmol/mg total protein for RAR beta and RXR gamma to 50 fmol/mg for RXR alpha. Hep G2 and HL60 cells express RAR alpha and RXR alpha proteins at approximately 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (approximately 5 fmol/mg) in Hep G2 cells. MCF-7 cells in culture express RAR alpha (approximately 32 fmol/mg), RAR gamma (approximately 35 fmol/mg), and RXR alpha (approximately 60 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Correlation with hormone binding and effects of metabolism.

The binding affinities of 9-cis-retinoic acid (9-cis-RA) and all-trans-retinoic acid (t-RA) for retinoic acid receptors (RAR) alpha, beta, and gamma and for retinoid X receptors (RXR) alpha, beta, and gamma were determined using the recombinant receptor proteins and were compared with each hormone's ability to activate transcription through the receptors in mammalian and yeast cell systems. 9-cis-RA bound to both the RXRs (Kd values = 1.4-2.4 nM) and the RARs (Kd values = 0.2-0.8 nM). The ability of 9-cis-RA to bind to the RARs and RXRs correlated with its ability to produce similar transactivation profiles with these receptors in mammalian and yeast cell assays. t-RA bound to the RARs (Kd values = 0.2-0.4 nM) and activated transcription through the RARs in mammalian and yeast cells. In contrast, while t-RA did not bind to the RXRs, it did activate the RXRs, albeit less potently than 9-cis-RA, in mammalian cells. In yeast, however, the RXRs activated transcription only in the presence of 9-cis-RA, not with t-RA. While RAR gamma is activated in yeast by either t-RA or 9-cis-RA, the overall level of transcription was increased upon the addition of hormone-occupied RXR. Metabolism studies suggest that while there was no cell-dependent interconversion between t-RA and 9-cis-RA in yeast, there was cell-dependent conversion of 9-cis-RA to t-RA in mammalian cells [corrected].