St John's wort (SJW), an extract of the medicinal plant Hypericum perforatum, is widely used as a herbal antidepressant. Recently, this agent has been found to adversely affect the metabolism of various coadministered drugs. Steroid X receptor (SXR), an orphan nuclear receptor, induces hepatic cytochrome P450 gene expression in response to diverse endogenous steroids, xenobiotics and drugs. Here, we report that, when coexpressed with SXR, a reporter construct derived from the cytochrome P450 3A promoter is activated by St John's wort. A GAL4-SXR ligand binding domain (LBD) fusion mediates concentration-dependent transactivation by SJW, whereas a mutant GAL4-SXR fusion, containing substitutions in key residues in a transactivation domain, is inactive. SJW recruits steroid receptor coactivator-1 to SXR in a two-hybrid assay and competes with radiolabelled ligand in binding studies, suggesting it interacts directly with the receptor LBD. Of two constituents of SJW, we find that hyperforin, but not hypericin, mediates both transactivation and coactivator recruitment by SXR. Our observations suggest that SXR activation by St John's wort mediates its adverse interaction with drugs metabolised via the CYP 3A pathway. Future development of SJW derivatives lacking SXR activation, may enable its antidepressant and drug-metabolising properties to be dissociated.

Progesterone, via its nuclear receptor, is mandatory not only for the induction and specification of mammary gland ductal side-branching and lobuloalveologenesis but also for carcinogen-induced mammary tumorigenesis. Notwithstanding these recent advances, a more comprehensive molecular explanation of progesterone-induced mammary morphogenesis is contingent upon the identification and characterization of mammary molecular targets that are responsive to the progesterone signal. Toward this goal, we report that calcitonin, a 32 amino acid peptide hormone involved in calcium homeostasis, is exclusively expressed in, and secreted from, luminal epithelial cells within the mammary gland of the pregnant mouse, and, importantly, its expression is progesterone-dependent. Conversely, the calcitonin receptor is present during all stages of post-natal mammary development examined, is localized to the myoepithelial cell lineage, and is not regulated by progesterone. Because calcitonin induction spatiotemporally correlates with increases in progesterone-induced mammary gland proliferation and structural remodeling, we posit that calcitonin - through its receptor - may be involved in one or both of these progesterone-dependent processes.

Interrelationships between thyroid hormone and estrogen actions have been documented with regard to a variety of physiological functions. Both hormones stimulate transcription of target genes by binding to their nuclear receptors that interact with specific responsive elements (estrogen and thyroid hormone response elements, i.e ERE and TRE, respectively) in the regulatory regions of the gene. In vitro studies have suggested that interplay between the two hormones might be due to cross-talk at hormone responsive elements, with the respective hormone receptors and ligands able to interact, although physiological relevance has yet to be proved. We have proposed a simpler mechanism for thyroid hormone effects on estrogen responses via increase in estrogen receptor alpha (ERalpha) with resultant increase in progesterone receptors, prolactin production and tumor growth. A pituitary cell line, GH3, has been widely used to investigate the function of mammo-somatotropic cells, especially regarding regulation of GH and prolactin production. In the present study, an ERE-luc reporter was transfected into GH3 cells and the responses to endogenous ERalpha were examined. We demonstrated that: (1)l -3,5,3'-triiodothyronine (T3) induces mRNA expression of ERalpha; (2) T3 alone is able to induce ERE-luc activity and this is inhibited by OH-tamoxifen; (3) T3 synergistically acts on estradiol (E2)-induced ERE responses; and (4) ERE-luc activity is enchanted by co-transfection of an ERalpha expression vector. These results support the hypothesis that estrogen responses are potentiated by T3 through up-regulation of ERalpha levels.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARalpha is highly expressed in liver, skeletal muscle, kidney, heart and the vascular wall. PPARgamma is predominantly detected in adipose tissue, intestine and macrophages. PPARs are activated by fatty-acid derivatives and pharmacological agents such as fibrates and glitazones which are specific for PPARalpha and PPARgamma respectively. PPARs regulate lipid and lipoprotein metabolism, glucose homeostasis, cell proliferation and differentiation, and apoptosis. PPARalpha controls intra- and extracellular lipid metabolisms whereas PPARgamma triggers adipocyte differentiation and promotes lipid storage. In addition, PPARs also modulate the inflammatory response. PPAR activators have been shown to exert anti-inflammatory activities in various cell types by inhibiting the expression of proinflammatory genes such as cytokines, metalloproteases and acute-phase proteins. PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the AP-1, nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription and nuclear factor of activated T-cells signalling pathways and by stimulating the catabolism of proinflammatory eicosanoids. These recent findings indicate a modulatory role for PPARs in inflammation with potential therapeutical applications in chronic inflammatory diseases.

Leptin, the product of the ob gene, is secreted into the circulation by white adipose tissue; its major role being to participate in the regulation of energy homeostasis. Plasma leptin levels are mainly determined by the relative adiposity of the subject; however, the great dispersion of values for any given body mass index and the noteworthy gender-based differences indicate that other factors are operating. Steroid hormones actively participate in the regulation of leptin secretion; however, non-steroid nuclear hormones have either not been studied or have provided contradictory results. In order to understand the role of hormones of the non-steroid superfamily such as 3,5,3'-tri-iodothyronine (T(3)), vitamin D(3) and retinoic acid (RA) in the control of leptin secretion, in the present work doses of 10(-9), 10(-8) and 10(-7) M of these compounds have been studied on in vitro leptin secretion. The organ culture was performed with omental adipose tissue samples from healthy donors (n=28). T(3) was devoid of effect at any dose studied, while an inhibition of leptin secretion was observed with 9-cis-RA (slight) and all-trans-RA (potent). Interestingly, vitamin D(3) exerted a powerfully inhibitory role at the doses studied, and its action was synergistic with all-trans-RA. In conclusion, in vitro leptin secretion by human adipose tissue is negatively controlled by either RA or vitamin D(3). The clinical significance of leptin regulation by this superfamily of nuclear receptors remains to be ascertained.

SUMMARY

After administration of oestradiol-17β to intact mature and immature rats, a decrease in the testicular concentration of specific oestradiol-binding sites was observed within 1 h. The binding capacity was replenished starting about 3 h after oestradiol administration and after 5 h the oestrogen receptor level had returned to control values. Exposure of intact animals to oestradiol-17β for longer periods (up to 24 h) did not result in an increase of receptor levels in testicular cytosol.

Mature animals which were hypophysectomized for periods of up to 10 days did not show a significant change in the number of specific oestradiol-binding sites in either total testicular tissue or dissected interstitial tissue. At 15 days or longer periods after hypophysectomy, an apparent increase in receptor concentrations in total testicular cytosol was observed due to a relative increase in the amount of interstitial tissue.

A specific oestradiol-binding protein is present in plasma of immature male rats aged less than 30 days. This plasma protein could also be demonstrated in the cytosol of testes of immature rats. In contrast to the cytosol receptor, which shows a moderate affinity for diethylstilboestrol (DES), the plasma protein did not bind DES. The sedimentation values of the plasma protein and the oestradiol receptor were 4 S and 8 S respectively. These differences in characteristics made it possible to demonstrate the presence of the oestradiol receptor in addition to the binding protein in testicular cytosol of rats from 14 days of age onwards. The nuclear receptor for oestradiol-17β could be demonstrated after incubation of testicular tissue of rats from 4 days of age onwards.

Abstract

In order to study whether peripheral action of thyroid hormones is altered in insulin deficiency and to elucidate the biological consequences of alteration of the cytosolic 3,5,3′-tri-iodo-l-thyronine (T₃) binding protein (CTBP), we measured malic enzyme, T₃-responsive nuclear n protein, CTBP and nuclear thyroid hormone receptor in the liver and kidney of streptozotocin (STZ)-induced diabetic rats that were treated with or without insulin and/or a receptor-saturating dose of T₃. The following results were obtained. 1. Induction of malic enzyme by T₃ was apparently diminished in diabetic rats. However, supplementary injection of insulin enabled previously given T₃ to take effect in diabetic rats. 2. T₃-responsiveness of other hepatic proteins (n protein and CTBP) was not altered by insulin in diabetic rats. 3. The level of n protein was increased by insulin in diabetic rats in vivo and in perfused rat liver, indicating that the hepatic n protein is a novel insulin-responsive protein. T₃ and insulin increased the level of n protein non-synergistically in diabetic rat liver. 4. Hepatic nuclear receptor levels were not altered in diabetic rats. 5. Hepatic CTBP levels were decreased in diabetic rats. This was not due to the toxic effect of STZ. Low CTBP level was only partially increased by insulin after 30 days of diabetic period. Renal CTBP levels were not altered in diabetic rats with or without insulin treatment. These results indicate that reduction of CTBP did not influence the hepatic response to a receptor-saturating dose of T₃, although CTBP may regulate the nuclear T₃ transport, and that fundamental action of a receptor-saturating dose of T₃ was not attenuated in diabetic rat liver.

Journal of Endocrinology (1994) 143, 55–63

ABSTRACT

To establish an artificial model for the study of decidual cell reaction in the rhesus monkey, long-term ovariectomized animals were treated with oestrogen followed by progesterone and an artificial deciduogenic stimulus was applied on day 16 of the treatment cycle. Histological examinations of the endometrial samples on different days following stimulation showed prominent epithelial plaque reaction by day 20. Marked subepithelial oedema surrounding plaque cells was also noted. On day 32, degenerating plaque cells with leucocytic infiltration were found. Beginning on day 24 of hormone treatment, stromal fibroblasts adjacent to plaque and glands showed swelling with rounding-up and enlargement of nuclei; these stromal cells appeared more decidual-like with moderate amounts of hypertrophy and few were binucleate by day 28. Endometrial granular cells became more conspicuous and appeared to increase in numbers adjacent to glands and blood vessels. Endometrial glands showed no notable changes, and were mostly tortuous with columnar epithelium and apocrine-type secretions. Around days 40 and 48 of the treatment cycle, the luminal margins of the glands became bosselated, and significant amounts of secretion were present in the glandular lumen. Measurement of immunoreactive concentrations of oestradiol-17β and progesterone in endometria from traumatized, hormone-treated monkeys revealed significant (P < 0·01) decreases in levels of oestradiol with time while the level of progesterone remained unchanged (P > 0·02). The corresponding profiles of concentrations of nuclear receptors for oestrogen and progesterone reflected the status of ligand availability in the endometrium; nuclear levels of progesterone receptors were maintained but significant decreases in cytoplasmic progesterone receptors (P < 0·03) and nuclear oestrogen receptors (P < 0·05) were evident with the period of treatment.

Journal of Endocrinology (1989) 120, 51–58

ABSTRACT

There is sexual dimorphism of specific species of mRNA in the neonatal rat brain and this sexual dimorphism may be imprinted by steroids of testicular origin during the perinatal period. According to current theories, only aromatizable androgens may cause sexual differentiation of sexual behaviour and function in the adult. The effects of oestradiol benzoate on mRNA synthesis in the neonatal female limbic system were therefore studied. In addition, cytosolic and nuclear oestrogen receptors were measured after administration of testosterone propionate, oestradiol benzoate or dihydrotestosterone (DHT). An attempt was made to distinguish between the brain oestrogen receptor and the plasma oestrogen-binding protein, alphafoetoprotein (AFP) by isoelectric focussing. After injection of 50 μg oestradiol benzoate s.c. to neonatal female rats, the expression of mRNA coding for sexually dimorphic proteins appeared to be changed to a male-type pattern. The overall density of labelling was noticeably greater and specific changes in labelled proteins were observed. These effects were observed within 3 h of injection. Both testosterone and oestradiol caused a marked depletion of cytosolic oestrogen receptors in the limbic system whereas DHT was ineffective in this respect. Nuclear receptors were present in equal abundance in male- and female-derived nuclei and only oestradiol was able to cause a significant (P < 0·025) increase in nuclear oestrogen receptors. The receptor and AFP could be distinguished by isoelectric focussing, since the pI of the receptor was 7·05, while that of AFP was 4·5. These results are consistent with the possibility that oestradiol alters transcription in the neonatal rat brain and may do this through the oestrogen receptor. Nevertheless, it is also possible that oestradiol could alter post-transcriptional events such as the stability of mRNA or the binding of tRNA to the polysomal complex.

Journal of Endocrinology (1989) 120, 83–88

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.