Nuclear Receptor Levels Research Articles

Differences in Cytochrome P450 and Nuclear Receptor mRNA Levels in Liver and Small Intestines between SD and DA Rats

This study aimed to clarify the differences in mRNA levels of cytochrome P450 (CYP) isoforms and nuclear receptors between Dark Agouti (DA) and Sprague-Dawley (SD) rats which are animal models for poor metabolizers and extensive metabolizers for CYP2D6, respectively. Using liver and small intestine tissues of both rat strains, we investigated the mRNA levels of CYP1A, 2A, 2B, 2C, 2D, 2E, and 3A subfamilies and nuclear receptors which regulate the transcription of CYP isoforms. In the liver, male DA rats showed a low CYP2D2 mRNA level but high mRNA levels of CYP3A1, 3A2, and 1A1 compared to SD rats. No significant difference was noted in other CYP isoforms. The mRNA levels of CAR were higher in DA rats than those in SD rats. In small intestine, the mRNA levels of CYP isoforms and nuclear receptors exhibited no significant strain differences. In addition, the activity of CYP3A in small intestinal microsome did not differ between SD and DA rats. Female DA rats exhibited higher mRNA levels of CYP3A1, 3A2, and 2B1 in the liver than female SD rats. In conclusion, the mRNA levels of CYP3A1 and 3A2 isoforms and CAR in the liver but not in the small intestines were different between DA and SD rats in both sexes.

Multiple Interactions between Peroxisome Proliferators‐Activated Receptors and the Ubiquitin‐Proteasome System and Implications for Cancer Pathogenesis

The peroxisome proliferator-activated receptors (PPAR) α, β/δ, and γ are ligand-activated nuclear receptors involved in a number of physiological processes, including lipid and glucose homeostasis, inflammation, cell growth, differentiation, and death. PPAR agonists are used in the treatment of human diseases, like type 2 diabetes and dyslipidemia, and PPARs appear as promising therapeutic targets in other conditions, including cancer. A better understanding of the functions and regulation of PPARs in normal and pathological processes is of primary importance to devise appropriate therapeutic strategies. The ubiquitin-proteasome system (UPS) plays an important role in controlling level and activity of many nuclear receptors and transcription factors. PPARs are subjected to UPS-dependent regulation. Interestingly, the three PPAR isotypes are differentially regulated by the UPS in response to ligand-dependent activation, a phenomenon that may be intrinsically connected to their distinct cellular functions and behaviors. In addition to their effects ongene expression, PPARs appear to affect protein levels and downstream pathways also by modulating the activity of the UPS in target-specific manners. Here we review the current knowledge of the interactions between the UPS and PPARs in light of the potential implications for their effects on cell fate and tumorigenesis.

Rexinoids and Breast Cancer Prevention

In this issue of Clinical Cancer Research , Li et al. investigate the chemopreventive potential of the retinoid, LG100268 ([1][1]). The application of retinoids to cancer chemoprevention has a long history which predates the identification of retinoid receptors ([2][2], [3][3]). Early clinical

Feedback regulation of thyrotropin-releasing hormone gene expression by thyroid hormone in the hypothalamic paraventricular nucleus.

Hypothyroidism caused by chemical or surgical thyroidectomy or hypophysectomy causes a substantial increase in the content of thyrotropin-releasing hormone (TRH) mRNA and proTRH exclusively in cells of the medial and periventricular paravocellular subdivisions of the hypothalamic paraventricular nucleus (PVN). This response may be important to raise the anterior pituitary thyrostat to promote increased secretion of thyroid-stimulating hormone (TSH) and to induce the secretion of a more biologically active TSH. The increase in TRH mRNA can be obliterated by stereotaxic implants of hormonally active L-triiodothyronine (T3) placed into the anterior hypothalamus but not by implants of the hormonally inactive 3,5'-diiodo-L-thyronine (T2); we therefore suggested that T3 has a direct action on TRH-containing cells of the PVN. Ablation of brainstem catecholaminergic projection fields to the PVN (known to stimulate TRH secretion) has no effect on TRH mRNA expression; beta 1 thyroid hormone receptor mRNA is present in extracts of the PVN. Euthyroid levels of serum T3 in hypothyroid animals achieved via intraperitoneally implanted osmotic minipumps are not associated with a return of PVN levels of TRH mRNA to normal unless circulating T3 levels are raised into the hyperthyroid range (1.7 times normal). This requirement is similar to that needed to normalize nuclear thyroid hormone receptor levels in the anterior pituitary of hypothyroid animals, suggesting that in addition to circulating T3 monodeiodination of T4 to T3 within the brain must also contribute to feedback inhibition of TRH mRNA. As Type II deiodinase activity is absent or very low in the PVN and does not rise with hypothyroidism, we propose that an alternative source for T4 monodeiodination exists within the central nervous system.

Age‐related changes in bile acid synthesis and hepatic nuclear receptor expression

Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.

Effects of budesonide on P‐glycoprotein expression in intestinal cell lines

P-glycoprotein (P-gp) is an important efflux transporter that supports the barrier function of the gut against invading antigens and against administered drugs. Since glucocorticoids, such as budesonide, are frequently used during inflammatory bowel disease we investigated how budesonide influences P-gp expression in different intestinal cell lines. LS180 and Caco-2 cells were incubated with budesonide and changes in P-gp expression were determined on mRNA, protein and functional level. The mRNA expression levels of glucocorticoid receptor (GR) and pregnane X receptor (PXR) were determined in these cell lines. PXR receptor was transiently transfected into Caco-2 cells. Budesonide showed an induction of P-gp in LS180 cells and a down-regulation in Caco-2 cells. Expression levels of nuclear receptors revealed high expression of PXR only in LS180 cells and exclusive expression of GR in Caco-2 cells. Mifepristone, an anti-glucocorticoid, could not reverse the down-regulation of P-gp by budesonide in Caco-2 cells. In PXR-transfected Caco-2 cells the budesonide-mediated down-regulation of P-gp was abolished. Furthermore the expression of cytochrome P450 3A4 (CYP3A4), another PXR target gene, was induced in PXR-transfected Caco-2 cells after budesonide treatment. Budesonide has the potential to influence MDR1 expression in vitro. In LS180 cells, the induction of MDR1 by budesonide probably is mediated via PXR. The mechanism of the down-regulation in Caco-2 cells still remains unclear, but GR does not seem to be involved. Further studies are required to evaluate how budesonide alters P-gp expression in vivo.

Tumor necrosis factor and interleukin 1 decrease RXR α, PPAR α, PPAR γ, LXR α, and the coactivators SRC-1, PGC-1 α, and PGC-1 β in liver cells

During the acute phase response, cytokines induce marked alterations in lipid metabolism including an increase in serum triglyceride levels and a decrease in hepatic fatty acid oxidation, in bile acid synthesis, and in high-density lipoprotein levels. Here we demonstrate that tumor necrosis factor (TNF) and interleukin 1 (IL-1), but not IL-6, decrease the expression of retinoid X receptor α (RXR α), peroxisome proliferator–activated receptor α (PPAR α), PPAR γ, liver X receptor α (LXR α), and coactivators PPAR γ coactivator 1 α (PGC-1 α), PGC-1 β, and steroid receptor coactivator 1 (SRC-1) in Hep3B human hepatoma cells. In addition, treatment of mice with TNF and IL-1 also decreased RXR α, PPAR α, PPAR γ, LXR α, and PGC-1 α messenger RNA (mRNA) levels in the liver. These decreases were accompanied by reduced binding of nuclear extracts to RXR, PPAR, and LXR response elements and decreased luciferase activity driven by PPAR and LXR response elements. In addition, the mRNA levels of proteins regulated by PPAR α (carnitine palmitoyltransferase 1 α) and LXR (sterol regulatory element binding protein) were decreased in Hep3B cells treated with TNF or IL-1. Finally, using constructs of the LXR α promoter or the PGC-1 α promoter linked to luciferase, we were able to demonstrate that a decrease in transcription contributes to the reduction in mRNA levels of nuclear hormone receptors and coactivators. Thus, our results suggest that decreased expression of nuclear hormone receptors RXR α, PPAR α, PPAR γ, and LXR α, as well as coactivators PGC-1 α, PGC-1 β, and SRC-1 may contribute to the cytokine-induced alterations in hepatic lipid metabolism during the acute phase response.

Estradiol‐induced modulation of estrogen receptor‐β and GABA within the adult neocortex: A potential transsynaptic mechanism for estrogen modulation of BDNF

Estrogen influences brain-derived neurotrophic factor (BDNF) expression in the neocortex. However, BDNF-producing cortical neurons do not express detectable levels of nuclear estrogen receptors; instead, the most abundant cortical nuclear estrogen receptor, ER-beta, is present in GABAergic neurons, prompting us to test the hypothesis that estrogen effects on BDNF are mediated via cortical inhibitory interneurons. Adult female ovariectomized rats were provided acute estrogen replacement and the number of cortical GABA, ER-beta, and ER-beta/GABA double-labeled neurons was examined. Within 48 hours of injection of 17-beta-estradiol, the number of perirhinal neurons double-labeled for ER-beta/GABA was reduced by 28% (P<0.01 compared to vehicle-treated ovariectomized controls), and all cells expressing detectable levels of GABA were reduced by 19% (P<0.01). To investigate potential relationships between estrogen receptors, GABAergic neurons, and BDNF-expressing cells, brain sections were double- or triple-labeled for ER-beta, GABAergic, and BDNF immunomarkers. The findings indicated that ER-beta-bearing inhibitory neurons project onto other GABAergic neurons that lack nuclear estrogen receptors; these inhibitory neurons in turn innervate BDNF-expressing excitatory cells. High estrogen states reduce cortical GABA levels, presumably releasing inhibition on BDNF-expressing neurons. This identifies a putative two-step transsynaptic mechanism whereby estrogen availability modulates expression of inhibitory transmitters, resulting in increased BDNF expression.

17β‐estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL‐68 cell cultures

Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17beta-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. HepG2 cells were exposed for 24 h to DCA (350 micromol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 micromol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 micromol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1-10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. 17Beta-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification.

Inhibition of NF-kappa B binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes.

Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-kappaB in monocytes in the presence of ongoing IkappaBalpha degradation, suggesting regulation of NF-kappaB activation downstream of IkappaBalpha degradation. DNA binding of NF-kappaB has been suggested to be regulated by other nuclear regulatory factors, including the glucocorticoid receptor (GR). Here, we show for the first time that acute alcohol (25 mM) exposure modulates GR activation in monocytes. Human peripheral blood monocytes were treated with lipopolysaccharide (LPS) in the presence or absence of alcohol (25 mM) for 1 hour. Nuclear GR levels were estimated by Western blotting and NFkappaB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 microM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IkappaBalpha mRNA known to be induced by GR/GRE activation was studied in total RNA extracts by the SuperArray method (SuperArray Inc., Bethesda, MD). LPS is a potent inducer of GR nuclear translocation and GR binding to the glucocorticoid response element (GRE). Acute alcohol treatment both induced (p < 0.05) and augmented (p < 0.05) LPS-stimulated GR nuclear levels. However, alcohol inhibits LPS-induced (nonligand bound) GR/GRE binding activity in monocytes. This inhibition of GR transactivation by alcohol was further confirmed by decreased expression (40%) of a target gene, IkappaBalpha. Thus, alcohol treatment increases nonligand-bound nuclear GR, but inhibits its transactivation function. Ligand-induced GR/GRE binding was decreased in alcohol-treated monocytes. Inhibition of ligand-induced GR/GRE binding by alcohol exposure is likely due to cytoplasmic retention of the GR. Our results show that acute alcohol exposure inhibits GR in monocytes by differently affecting ligand- and nonligand-induced GR nuclear translocation. These data also suggest that acute alcohol regulates GR activation in monocytes concomitant to inhibition of NF-kappaB activation.

Estradiol/Androgen Receptors During Aging: Microsomal Distribution in Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor lev...

Immunochemical characterization of human estrogen sulfotransferase SULT1E1 in human embryonic kidney 293 cells

Human sulfotransferase SULT1E1 is the major cytosolic sulfotransferase isoform that catalyzes the sulfation of estrogens. This reaction increases the water solubility of the estrogen molecule, which may lead to altered cellular estrogen distribution and, as a result, may affect the cellular response to estrogenic signaling. To date, little is known about the role(s) of SULT1E1-catalyzed estrogen sulfation in the regulation of intracellular estrogen-activated signaling processes. In the current studies, the expression of SULT1E1 in human embryonic kidney 293 (HEK293) cell line was determined using immunocytochemistry and green fluorescent protein (GFP)-tagging methods. The results showed that immunoreactivity to anti-SULT1E1 antibody was highly detectable in the cytoplasm of HEK293 cells. The GFP-SULT1E1 fusion protein also had the same cytosolic location as the native protein. Additional studies using immunoblot analysis were not able to detect the expression levels of human nuclear estrogen receptors α and β in HEK293 cells. This is consistent with previous results (Thomas et. al., 2005) and suggests that the physiological role(s) of SULT1E1-catalyzed sulfation reactions in HEK293 cells are not associated with estrogen receptors α and β coupled cellular signaling pathways.

Decreased hepatic expression of PPAR‐γ coactivator‐1 in cholesterol cholelithiasis

Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.

Steroid hormone receptors and coregulators in endocrine‐resistant and estrogen‐independent breast cancer cells

Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ERbeta upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ERalpha levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy.

Expression and Regulation of Progestin Membrane Receptors in the Rat Corpus Luteum

Despite evidence strongly supporting progesterone's autocrine actions in the rat corpus luteum (CL), classical progesterone receptors (PR) have not been detected in this gland. Alternatively, in several other systems, progestins have been reported to activate nongenomic pathways via putative progestin membrane receptors (PMRs). The aim of this investigation was to determine whether rat CL membranes bind progestins and contain PMR homologs and whether these proteins are expressed during CL development in a manner that parallels luteal function. We found that luteal cell membranes specifically bind progesterone. Low levels of progesterone and 20alpha-dihydroprogesterone decreased binding of [(3)H]progesterone, whereas androstenedione, 17alpha-hydroxyprogesterone, and pregnenolone were less potent. Other steroids, including corticosterone, mifepristone, and estradiol, were ineffective. We found that the rat CL expresses five genes previously postulated to encode for putative PMRs: PMRalpha, PMRbeta, PMRgamma, PR membrane component 1 (PRMC1), and Rda288. Pmralpha, Pmrgamma, and Prmc1 transcripts rose steadily during pregnancy whereas Pmrbeta and Rda288 remained constant. Just before parturition, concomitant with falling progesterone levels, Pmralpha, Pmrbeta, and Prmc1 decreased. Luteal PMRalpha and PRMC1 protein levels were lower in samples taken at the end of pregnancy compared with midpregnancy samples. Ergocriptine, which inhibits the secretion of prolactin, the primary luteotrophic hormone in the rat CL, reduced Pmralpha, Pmrbeta, and Prmc1 expression significantly. Ergocriptine effects were prevented by coadministration of prolactin. These findings provide evidence for the expression and regulation of putative membrane-bound progestin-binding proteins in the rat CL, a tissue that does not express detectable levels of nuclear progesterone receptors.

Ovariectomy in Mice Decreases Lipid Metabolism-Related Gene Expression in Adipose Tissue and Skeletal Muscle with Increased Body Fat

Postmenopausal women as well as rodents after ovariectomy, which results in a lack of estrogen, can become obese. Ovariectomy-induced obesity in mice is associated with a decrease in oxygen consumption, indicating repressed energy expenditure. In this study, to elucidate the mechanism of weight gain after ovariectomy, we examined the expression patterns of genes related to energy expenditure and lipid metabolism, in mouse tissues including adipose tissue and skeletal muscle. In adipose tissue and skeletal muscle, at 2-4 wk after ovariectomy, levels of nuclear receptors and cofactors involved in energy expenditure such as ERR1, PPARalpha and PPARdelta, and PGC1alpha and PGC1beta were lower than in control mice. mRNA levels of their targets, medium-chain acyl coenzyme A dehydrogenase and acetyl CoA oxidase, enzymes for fatty acid beta-oxidation, were lower. In addition, the expression of PPARgamma and SREBP1, transcription factors important for lipogenesis, was decreased, as well as that of acetyl CoA carboxylase and fatty acid synthase, enzymes for fatty acid synthesis, and diacyl glycerol acetyl transferase 1 and 2, enzymes for triglyceride synthesis. These changes in gene expression are consistent with the obese phenotype in mice after ovariectomy. Thus a decrease in the expression of energy expenditure-related genes in adipose tissue and skeletal muscle could, in part, be responsible for obesity after ovariectomy.

Estradiol/Androgen Receptors During Aging: Microsomal Distribution In Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor levels (mean percentage +/- 50%). These findings suggest that the biological effects due to the subcellular distribution of AR or ER and that their microsomal distribution would constitute a reserve mechanism and activation of receptors for the continued growth of benign prostatic hypertrophy.

Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.

Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4 alpha (HNF4 alpha, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4 alpha. PXR also interacted with peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha), which interacted with HNF4 alpha and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4 alpha and reduced PGC-1 alpha interaction with HNF4 alpha. Chromatin immunoprecipitation assay showed that PXR, HNF4 alpha, and PGC-1 alpha bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1 alpha from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4 alpha and blocks PGC-1 alpha activation with HNF4 alpha and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.

Developmental expression and hormonal regulation of glucocorticoid and thyroid hormone receptors during metamorphosis in Xenopus laevis.

Corticosteroids, the primary circulating vertebrate stress hormones, are known to potentiate the actions of thyroid hormone in amphibian metamorphosis. Environmental modulation of the production of stress hormones may be one way that tadpoles respond to variation in their larval habitat, and thus control the timing of metamorphosis. Thyroid hormone and corticosteroids act through structurally similar nuclear receptors, and interactions at the transcriptional level could lead to regulation of common pathways controlling metamorphosis. To better understand the roles of corticosteroids in amphibian metamorphosis we analyzed the developmental and hormone-dependent expression of glucocorticoid receptor (GR) mRNA in the brain (diencephalon), intestine and tail of Xenopus laevis tadpoles. We compared the expression patterns of GR with expression of thyroid hormone receptor beta (TRbeta). In an effort to determine the relationship between nuclear hormone receptor expression and levels of ligand, we also analyzed changes in whole-body content of 3,5,3'-triiodothyronine (T(3)), thyroxine, and corticosterone (CORT). GR transcripts of 8, 4 and 2 kb were detected in all tadpole tissues, but only the 4 and 2 kb transcripts could be detected in embryos. The level of GR mRNA was low during premetamorphosis in the brain but increased significantly during prometamorphosis, remained at a constant level throughout metamorphosis, and increased to its highest level in the juvenile frog. GR mRNA level in the intestine remained relatively constant, but increased in the tail throughout metamorphosis, reaching a maximum at metamorphic climax. The level of GR mRNA was increased by treatment with CORT in the intestine but not in the brain or tail. TRbeta mRNA level increased in the brain, intestine and tail during metamorphosis and was induced by treatment with T(3). Analysis of possible crossregulatory relationships between GRs and TRs showed that GR mRNA was upregulated by exogenous T(3) (50 nM) in the tail but downregulated in the brain of premetamorphic tadpoles. Exogenous CORT (100 nM) upregulated TRbeta mRNA in the intestine. Our findings provide evidence for tissue-specific positive, negative and crossregulation of nuclear hormone receptors during metamorphosis of X. laevis. The synergy of CORT with T(3) on tadpole tail resorption may depend on the accelerated accumulation of GR transcripts in this tissue during metamorphosis, which may be driven by rising plasma thyroid hormone titers.

Expression of neurogranin and neuromodulin is affected in the striatum of vitamin A-deprived rats

Our previous data showed that vitamin A deficiency (VAD) induces, in whole brain, a reduced amount of mRNA for brain retinoic acid (RA) and triiodothyronine (T3) nuclear receptors (i.e., RAR, RXR, and TR, respectively), which is accompanied by reduced amounts of mRNA and protein of neurogranin (RC3, a neuronal protein involved in synaptic plasticity) as well as selective behavioral impairment. Given the important role of retinoids for optimal brain functioning, the effects of vitamin A depletion and subsequent administration of RA or T3 on the mRNA levels of RA and T3 nuclear receptors and on two target genes' ( RC3 and neuromodulin or GAP43) mRNA and protein levels were examined in the hippocampus, striatum, and cerebral cortex. A quantitative real-time polymerase chain reaction (PCR), in situ hybridization, and Western blot analysis demonstrated that the striatal region is the brain site where both RA and T3 signaling pathways are most affected by VAD. Indeed, rats fed a vitamin A-free diet for 10 weeks exhibited decreased expression of RAR, RXR, TR, RC3, and GAP43 in the striatum. The administration of T3 to these vitamin A-deprived rats reversed the reduction in mRNA levels of RA and T3 nuclear receptors and in mRNA and protein levels of target genes in this region. These data suggest that modifications that appear preferentially in the striatum, a region highly sensitive to vitamin A bioavailability, may contribute to neurobiological alterations and the spatial learning impairment that occurs in vitamin A-deprived animals.