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B. D. Greenstein


Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0·4 m-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed.

J. Endocr. (1984) 102, 181–188

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T Takeda, H Kurachi, T Yamamoto, Y Nishio, Y Nakatsuji, K Morishige, A Miyake, and Y Murata

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

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M Takamoto, K Tsuji, T Yamashita, H Sasaki, T Yano, Y Taketani, T Komori, A Nifuji, and M Noda

Hedgehog signaling is considered to play a crucial role in chondrogenesis by regulation through a network of cytokine actions, which is not fully understood. We examined the effect of hedgehog signaling on the expression of core-binding factor a1 (Cbfa1), a critical transcription factor for the development of bone and cartilage. Primary chondrocytes prepared from the costal cartilage of newborn mice were treated with N-terminal fragment of recombinant murine sonic hedgehog (rmShh-N). Northern blot analysis indicated that Cbfa1 mRNA expression levels in the chondrocyte cultures were elevated by the treatment with rmShh-N. rmShh-N treatment enhanced 1.8 kb Cbfa1 promoter activity in chondrocytes, suggesting the presence of transcriptional control. As Cbfa1-binding site(s) have been located in the promoter of the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) gene, we also examined RANKL expression. rmShh-N treatment upregulated RANKL and RANK mRNA expression levels in chondrocytes. Interestingly, RANKL suppressed the hedgehog enhancement of alkaline phosphatase activity in chondrocytes, suggesting the presence of a link between these signaling molecules. We conclude that hedgehog signaling activates Cbfa1 gene expression through its promoter in chondrocytes, and also activates and interacts with RANKL to maintain cartilage development.

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J Cheung, YT Mak, S Papaioannou, BA Evans, I Fogelman, and G Hampson

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E(2)) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E(2) and RAL at concentrations ranging from 10(-12) to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E(2) and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E(2) (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E(2) and RAL in the HCC1 cells (E(2) (10(-8), 10(-7) and 10(-6) M), % reduction (means+/-S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84+/-7.4, 70.8+/-2.9*, 78.2+/-4.8*; IL-1beta: 79+/-10, 72.8+/-8.2*, 66.6+/-2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4+/-5*, 79+/- 5.2*, 102+/-7.7; IL-1beta: 67.9+/-3.2*, 69+/-2.5*, 73.8+/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E(2) and RAL (% reduction E(2) (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5+/-8.4*, 80+/-6.7*, 62.8+/-8.9*; HCC1: 109+/-4, 98.8+/-6, 54.5+/-3.4*; RAL (10(-8), 10(-7) and 10(-6) M) - HOB: 81.5+/-5.5*, 62.7+/-7.4*, 55.2+/-10.9*; HCC1: 92.7+/-7.4, 67+/-12.2*, 39+/-4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E(2). In contrast, a significant reduction in RANKL was seen with RAL at 10(-7) and 10(-6) M (66+/-6.4% and 74+/-3% respectively). There was no change in OPG mRNA expression following E(2) or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10(-8), 10(-7)and 10(-6) M) treatment (% change from controls: 52+/-2*, 62+/-1*, 53+/-5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72+/-5.5, P<0.05). In addition, there was a significant decrease in the RANKL/OPG ratio after RAL at 10(-6) M (HOB: 65.6+/-5*, HCC1: 56.9+/-20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E(2). However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E(2). In conclusion, the study shows that RAL and E(2) have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E(2), RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.

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Binding of 125I-labelled bovine TSH to crude membrane fractions of human thyroid tissue was a saturable, hormonally specific process which yielded non-linear Scatchard plots with limiting affinities of approximately 109 and 107l/mol. Binding activity in membranes was soluble in Triton X-100, was inhibited specifically by immunoglobulins from patients with Graves's disease, and was increased by the beta-blocking drug, propranolol. In contrast, purified nuclear preparations showed a predominance of lower affinity binding, and their binding activity was insoluble in Triton and insensitive to immunoglobulins from patients with Graves's disease and to propranolol. Tryptic digestion liberated only low affinity binding activity from membranes or nuclei. It was concluded that human thyroid tissue contains independent classes of TSH-binding sites, which differ in their chemical, immunological and hormone-binding properties.

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Marcello Maggiolini and Didier Picard

β-estradiol (E 2 ), is no exception. Estrogens have a multitude of different molecular targets, including two well-characterized members of the nuclear receptor superfamily, the estrogen receptors (ER) α and β (listed as ESR1 and ESR2 in the HUGO

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G. Auf and R. Ghanadian

The demonstration and partial characterization of a high-affinity saturable binding component for oestradiol-17β in the cytosol and nuclear extract of the benign hypertrophied human prostate is reported. This binding component was found to precipitate with protamine sulphate and to exhibit marked specificity for oestradiol and diethyl-stilboestrol (DES) but not dihydrotestosterone (DHT) or progesterone. Analysis of oestradiol-labelled cytosol on low ionic strength glycerol gradients revealed a binding component with a sedimentation coefficient of 4S which was inhibited with DES but not DHT and destroyed either by preheating labelled cytosol at 45 °C for 30 min or by treatment with the sulphydryl blocking agent, parachloromercureobenzoate. The oestradiol-binding complex precipitated by ammonium sulphate was found to have an isoelectric point (pI) of 6·3 as analysed on polyacrylamide-gel plates. The oestradiol-binding component in the nuclear extract also exhibited similar steroid specificity and sedimented with a coefficient of 2·8S on high ionic strength glycerol gradients. Isoelectric focusing of the heparin-extracted nuclear oestradiol-binding complex revealed a pI of 6·0. The dissociation constants (K d) and the concentrations of the cytoplasmic and nuclear oestradiol-binding sites in 19 samples of benign hypertrophied prostates were estimated by Scatchard plot analyses. The mean K d and concentration of binding sites in the cytosol were 2·9 ± 2·6 (s.d.) nmol/l and 11·7± 8·4 fmol/mg protein respectively. The corresponding values for nuclear extract were 4·1 ±7·5 nmol/l and 347·1±415·5fmol/mg DNA respectively. There was no significant difference between the mean concentration of oestradiol-binding sites in the two cellular fractions.

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Cyril S Anyetei-Anum, Vincent R Roggero, and Lizabeth A Allison

, Mullur et al . 2014 , Mondal et al . 2016 , Mendoza & Hollenberg 2017 , van der Spek et al . 2017 ). Much of thyroid hormone action is mediated by the thyroid hormone receptors (TRs), members of the nuclear receptor superfamily that act as ligand

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R L Moore, Y Dai, and D V Faller

Introduction Steroid hormone receptors, such as the estrogen (ER) and androgen receptors (AR), as well as the progesterone (PR), glucocorticoid (GR), and mineralocorticoid receptors (MR) are part of a large nuclear receptor family of eukaryotic

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Kyuyong Han, Haengseok Song, Irene Moon, Robert Augustin, Kelle Moley, Melissa Rogers, and Hyunjung Lim

Introduction Retinoids, e.g. retinoic acids (RA), utilize two subfamilies of nuclear receptors, retinoic acid receptors (RARs), and retinoid X receptors (RXRs). Three subtypes of RAR, α, β, and γ, exist as receptors of trans - and 9