Takahisa Hirose

Cloning of a cDNA encoding the murine orphan receptor RZR/RORγ and characterization of its response element

In this study, we describe the cloning of the mouse homologue of the orphan receptor, RZR/ROR gamma, a member of the nuclear receptor superfamily, from a mouse muscle cDNA library. The amino acid sequence of mouse ROR gamma (mROR gamma) is highly homologous to that of human ROR gamma, with an overall identity of 88%. Northern blot analysis using RNA from different tissues showed that mROR gamma was found to be highly expressed in skeletal muscle, liver and kidney. Analysis of the ROR gamma-response element using in vitro synthesized ROR gamma revealed that it binds as a monomer to response elements composed of a single core motif GGTCA preceded by a 6 bp AT-rich sequence. The ROR gamma-binding specificity was further defined by mutational analysis of the consensus RORE. ROR gamma was able to activate RORE-dependent transcription of the CAT reporter gene in mouse fibroblast D1 cells. ROR alpha 1 and ROR gamma inhibit the transactivation induced by GAL4(DBD)-ROR gamma in fibroblast D1 cells suggesting that these receptors compete for binding to the same coactivators.

Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor

Estrogens are the most effective agents available for preventing osteoporosis, and their principal role in bone metabolism is the inhibition of interleukin-6 (IL-6) production in osteoblasts and bone marrow stromal cells. We examined the mechanism of inhibitory effect of estrogens on the 190 bp proximal promoter of the IL-6 gene. Promoter activity induced by transfection of both NF-kappaB p65 subunit and NF-IL6 was decreased by 45% by estradiol (E2)-estrogen receptor (ER) complexes. The inhibitory effect of E2 was also observed on a mutant IL-6 promoter in which the NF-IL6 binding site was disrupted. E2 repressed the wild-type promoter activity induced by NF-kappaB p65 subunit alone, but had no effect on that induced by NF-IL6 alone. These findings suggested that estrogens inhibit IL-6 production by interfering with the function of NF-kappaB rather than that of NF-IL6. The ER mutant, HE19, which does not contain the A/B domain, repressed the induction by NF-kappaB to the same extent as wild-type ER HE0, whereas the effect of C-terminal deletion mutant, HE21, was only marginal. The antiestrogen, 4-hydroxytamoxifen (OHT), had no effect on IL-6 promoter activity, suggesting that E2-induced conformational change of the hormone binding domain plays an important role in protein-protein interaction between ER and NF-kappaB. E2 had no effect on the nuclear translocation of NF-kappaB, and electrophoretic mobility shift assay showed that the presence of E2-ER complexes did not affect the ability of NF-kappaB to bind to specific DNA sequences.

Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis.

Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand‐activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3‐kb transcript generally expressed at higher levels than the larger 7.4‐ or 8.0‐kb transcript. In both the mouse and rat, the 2.3‐ and 7.4‐kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4‐kb mRNA appearing earlier during testicular development. The major 2.3‐kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady‐state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3′ termination site of the 2.3‐kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation. Mol. Reprod. Dev. 50:93–102, 1998.

Increased Interleukin-6 Production in Mouse Osteoblastic MC3T3-E1 Cells Expressing Activating Mutant of the Stimulatory G Protein

The McCune–Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café‐au‐lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the α subunit of the Gs protein (Gsα) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin‐6 (IL‐6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL‐6 production by activating mutant Gsα in bone cells, we established mouse osteoblastic MC3T3‐E1 cells stably transfected with the activating mutant Gsα expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL‐6 than the cells transfected with control vector or wild‐type Gsα expression vector. Analysis of the IL‐6 promoter revealed that any of the AP‐1, nuclear factor (NF)‐IL6, and NF‐κB binding elements are important for the activating mutant Gsα‐induced gene expression. Electrophoretic mobility‐shift assays using nuclear extracts of the mutant Gsα‐expressing cells showed that phospho(Ser133)‐cAMP‐responsive element binding protein (CREB), AP‐1, NF‐IL6, and NF‐κB were increased, compared with the control cells or the wild‐type Gsα‐expressing cells. These results indicate that activating mutant Gsα increases the transcriptional factors binding to CRE, AP‐1, NF‐IL6, and NF‐κB elements to induce IL‐6 gene expression in the osteoblastic cells.

A single nucleotide substitution in the D domain of estrogen receptor cDNA causes amino acid alteration from Glu-279 to Lys-279 in a murine transformed Leydig cell line (B-1 F).

A polymerase chain reaction method was carried out to address the possible presence of estrogen receptor (ER) mutations in murine transformed cell lines. The segment of ER cDNA coding the C and D domains was amplified and cloned into pUC 19. Mammary carcinoma cell line (SC-3) did not show any mutation in this segment. However, the sequence analysis of ER in the Leydig cell line (B-1 F) revealed a single base change at acidic Glu-279 (GAA) to basic Lys-279 (AAA) compared with murine uterus ER cDNA. The biological significance of this mutation is discussed.

Interaction of DP-TAT-59, an active metabolite of new triphenylethylene-derivative (TAT-59), with estrogen receptors

DP-TAT-59, (Z)-2-(4-(1-(4-hydroxyphenyl)-2-(4-isopropylphenyl)-1-butenyl) phenoxy)-N, N-dimethylethylamine, has been reported to inhibit estrogen-stimulated growth of MCF-7 cells as well as rat uterus at lower concentrations than the hydroxymetabolite of tamoxifen (4-OH-TAM). In the present study, the growth of mouse Leydig cell tumor, B-1F cells were also more effectively inhibited by DP-TAT-59 than 4-OH-TAM. Additionally, the expression of estrogen responsive element ligated CAT gene transfected into B-1F cells was also suppressed by DP-TAT-59. Thus, the interaction of DP-TAT-59 with estrogen receptor (ER) was characterized and compared with that of 4-OH-TAM using immature rat and bovine uteri. The dissociation constant of DP-TAT-59 to ER of immature rat uterus was 0.24 nM and was similar to that of 4-OH-TAM (Kd = 0.20 nM) and estradiol (Kd = 0.29 nM). Using sucrose density gradients, the sedimentation constant of DP-TAT-59 with bovine uterus was 4.9S, which was similar to that of estradiol (5.1S) and 4-OH-TAM (5.3S). However, the elution profile of the DP-TAT-59-ER complex from a DEAE-Sephadex column was different for both estradiol-and 4-OH-TAM-ER complexes. These results suggest that ER forms different complexes with DP-TAT-59 than estradiol or 4-OH-TAM, while the ER binding affinity of these compounds are similar to each other.

FUNCTIONAL ABNORMALITY OF GLUCOCORTICOID RECEPTOR IN SHIONOGI CARCINOMA 115 CELLS AS EVIDENCED BY GENE TRANSFER EXPERIMENTS

The assay systems for steroid receptor functions in steroid-sensitive cells (SC-3 cells) were developed in which hormone-responsive element linked to a reporter gene [chloramphenicol acetyl transferase (CAT) gene] was transfected by the electroporation technique. Stimulation with androgen of SC-3 cells transfected with mouse mammary tumor virus promoter-CAT gene (MMTV-CAT) resulted in clear enhancement of CAT activity, whereas glucocorticoid required abnormally high concentrations to obtain significant stimulation. The simultaneous addition of glucocorticoid surprisingly inhibited androgen-induced CAT activity in SC-3 cells, whereas glucocorticoid and androgen acted together synergistically to activate CAT activity in T 47D cells. When SC-3 cells were cotransfected with the expression vector of human glucocorticoid receptor (GR) gene, inhibition with glucocorticoid of androgen-enhanced CAT activity was abolished. These results would suggest that SC-3 cells contain functionally abnormal GR.

Non-classical androgenic actions of RU38486 in androgen-responsive shionogi carcinoma 115 cells in serum-free culture

Antiglucocorticoid and antiprogestin RU38486 (RU486) stimulated the growth of highly androgen- and moderately glucocorticoid-sensitive SC-3 cells (a cloned cell line from Shionogi mouse mammary carcinoma 115) in a dose-dependent manner. A maximal 8-fold stimulation of growth by RU486 has been observed at 10(-7) M in a serum-free medium and its potency has been found to be almost the same as that of dexamethasone (Dex). The growth rate of SC-3 cells treated by triamcinolone acetonide (TA) or Dex combined with RU486 at 10(-9)-10(-7) M was enhanced compared to cells treated by TA or Dex alone, indicating that RU486 had additive rather than antagonistic effects. Our previous study revealed that RU486 could compete with the specific uptake of [3H]testosterone in intact SC-3 cells at relatively low affinity and the present study showed that the stimulatory effect of RU486 on the growth of SC-3 cells was significantly inhibited by pure antiandrogen flutamine and that half-maximal inhibition by flutamine was achieved at 10(-6) M. Moreover, we demonstrated that the conditioned medium from RU486-stimulated SC-3 cells contained growth-promoting activity which caused a 3.5-fold increase in DNA synthesis by SC-3 cells in the absence of RU486 and which was abolished by treatment with heparin-Sepharose. These results indicate that RU486-induced growth of SC-3 cells may be expressed as an androgenic activity through androgen receptor and mediated by a heparin-binding growth factor.

Identification of unoccupied but transformed nuclear estrogen receptor in cultured mouse leydig cell

The molecular forms of estrogen receptor (ER) in estrogen-responsive mouse Leydig cell line (B-1) have been examined in relation to their biological activity. ER was predominantly recovered in the nuclear fraction upon homogenization even after cells were precultured in the absence of E2 and Phenol Red. This unoccupied nuclear ER (ERn) whose hormone binding ability was extremely thermostable could be extracted with 0.4 M KCl. This stability enabled us to determine hydrodynamic parameters in the ligand-free condition. The Stokes radius and sedimentation constant of this ERn in high salt condition were 5.5 nm and 6.0S, respectively, resulting in its molecular weight of 140,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ER labeled with [3H]tamoxifen aziridine gave a single band of 65,000 Da, indicating that this ERn had a oligomer structure similar to that of transformed nuclear ER complexed with estrogen in the putative target cells. Therefore, we further examined the possibility that this ERn in B-1 cells can activate estrogen-responsive genes without any aid from estrogen. Estrogen responsive element-thymidine kinase promoter-chloramphenicol acetyltransferase fusion gene (ERE-tk-CAT) was transfected into B-1 cells. CAT activity was enhanced only in cells stimulated with estrogen. It may be concluded from these results that transformed ERn can be formed in the absence of estrogen but that binding to estrogen may be required in order to exert its biological activity.

Antiplatelet agent cilostazol potentiates adipocyte differentiation of 3T3-L1 cells

Cilostazol is an antiplatelet drug, which has beneficial effects in treatment of intermittent claudication and decreases serum triacyiglycerol level in these patients. In this study, we examined adipogenic potency of cilostazol using 3T3-L1 preadipocyte cell line because cilostazol is one of the tissue specific phosphodiesterase (PDE) inhibitors. Addition of cilostazol into the differentiation medium including insulin and dexamethasone, induced the adipocyte differentiation without isobutyl methylxanthine (IBMX). Compared with the cells incubated with vehicle, the cells treated with cilostazol contain much more lipid droplets in the cells 6 days after induction of differentiation. Adipocyte specific gene like stearoyl-CoA desaturase was strongly induced after addition of cilostazol. C/EBPbeta, which is induced by IBMX was also induced by cilostazol. These findings suggest a possibility that adipogenic effect of cilostazol is one of the mechanisms, by which this agent decreases blood triacylglycerol level in the intermittent claudication patients.