Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Increase of estrogen receptor level by thyroxine in estrogen dependent pituitary tumor (MtT/F84) in rats

A rat transplantable pituitary tumor, MtT/F84, grows much faster in E2 treated rats than in normal females, but is much retarded in thyroidectomized rats. Triiodothyronine (T3) administration in a drinking water increased the tumor growth by the dose dependent manner. The tumor contained both estrogen receptor (ER) and T3 receptor. ER levels both in the nuclei and cytosols elevated 2 to 3 times by the T3 administration compared to those of control. E2 administration promotes the growth of MtT/F84 through elevation of nuclear ER level. T3 may directly elevate cellular ER level and thus it may enhance estrogenic actions including the tumor growth.     

Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.     

Estrogen target cells. Establishment of a cell line derived from the rat pituitary tumor MtT/F4.

Pretreatment of rabbit kidney cells with cytochalasins B and D (CB, CD) enhanced herpes simplex virus type 2 (HSV-2) DNA infectivity 3- to 6-fold over values obtained using the standard CaCl₂ technique. Cells were pretreated with CB for 4–6 h to achieve infectivity enhancement. A lower concentration of CD, and shorter pretreatment periods, resulted in comparable DNA infectivity. Separate exposure of cells to colchicine, colcemid, or vinblastine increased DNA infectivity 7-, 6-, and 5-fold, respectively, over control values. Additional enhancement was obtained when CD was used together with any one of the aforementioned drugs. Maximal enhancement of HSV-2 DNA infectivity was obtained by pretreating recipient cells with a drug mixture containing colchicine, colcemid, and CD. This treatment maximized infectivity levels 20- to 30-fold over CaCl₂ control values.     

Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor

The effects of 17 beta-estradiol (E2) on MtT/F4 pituitary tumor growth and on prolactin (PRL) and growth hormone mRNA expression were analyzed in F344 female rats. E2 (10 mg) stimulated pituitary PRL cell hyperplasia and PRL mRNA, but inhibited growth of the transplantable tumors. The expression of both PRL and growth hormone mRNA levels was increased in the MtT/F4 tumors. The effects of E2 on increasing PRL mRNA levels were more marked in the pituitary compared with the tumors. These results indicate that estrogens stimulate proliferation and PRL expression in the pituitary while inhibiting cell proliferation in the MtT/F4 tumor. E2 also stimulated both growth hormone and PRL mRNA expression in the MtT/F4 transplantable tumor.     

Global analysis of gene expression in the estrogen induced pituitary tumor of the F344 rat

The F344 rat rapidly forms large prolactinomas in response to chronic estrogen treatment. To identify genes expressed in the course of this estrogen induced pituitary tumor growth, we performed microarray analysis on the F344 rat pituitary after chronic estrogen treatment and on untreated controls. At a significance level set to minimize type I error, some 72 genes were found to be differentially expressed between estrogen treated and untreated. Of those genes, 70 have not been reported previously as being affected by estrogen in the F344 rat pituitary. Since many other investigators have studied the effect of estrogen on specific gene expression in rat pituitary, we also examined the mRNA expression of the 36 genes that have been previously reported as having their expression affected by estrogen in the rat pituitary. Of these, 13 were found to have their expression affected by estrogen treatment in the same direction as had been reported by others.     

Leukaemia inhibitory factor and interleukin 6 inhibit secretion of prolactin and growth hormone by rat pituitary MtT/SM cells.

The rat pituitary cell line, MtT/SM, has the characteristics of somatomammotrophs. The cells secrete both prolactin (PRL) and growth hormone (GH). We examined the effects of cytokines such as leukaemia inhibitory factor (LIF), interleukin 6 (IL-6), oncostatin M and interleukin 11 on the secretion of these hormones by the cells. These cytokines stimulate proliferation of the cells and inhibit the secretion of PRL by 70-80% and that of GH by 50%. They induce tyrosine phosphorylation of STAT3 in the cells. The cells containing PRL or GH decreased at 48 h after treatment of the cells with LIF or IL-6. These results suggest that the LIF/IL-6 family of cytokines inhibits the functions of mammotrophs and somatotrophs in the pituitary gland.     

MLT抑制E2诱发的大鼠垂体PRL瘤的增生涉及雌激素受体的作用

目的：探讨褪黑素（melatonin,MLT）抑制17-β-雌二醇（17-β-estradiol,E2）诱发垂体催乳素（prolactin,PRL）瘤的增生及其机制的初始阶段,MLT对雌激素受体的作用。方法：在体实验采用每日定时给各组SD大鼠分别皮下注射不同浓度的MLT,建立MLT抑制E2诱发的垂体PRL瘤增生的动物模型。离体实验采用原代培养细胞原位杂交方法,探讨垂体PRL瘤细胞MLT受体的表达、MLT对雌激素受体（ER）表达的作用;应用电泳迁移率改变（EMSA）的方法,观察MLT对雌激素受体（ER）与雌激素反应元件（ERE）结合的效应。结果：每只大鼠每日定时皮下注射0．25或0．50mg MLT能显著抑制E2诱发的垂体PRL瘤的增生（分别P〈0．01、P〈0．05）;F4诱发的垂体PRL瘤细胞内有MLT受体MLT1a和MLT1b;给0．25mg／day／rat MLT组的大鼠垂体PRL瘤细胞内ER的表达显著减少（P〈0．01）、ER与ERE的结合量显著降低（P〈0．01）。结论：一定剂量的MLT能显著抑制E2诱发的SD大鼠垂体PRL瘤的增生,其作用机制之一可能与MLT抑制ER的表达、及其部分阻断ER与ERE的结合有关。     

Increased peptidylarginine deiminase expression during induction of prolactin biosynthesis in a growth-hormone-producing rat pituitary cell line, MtT/S.

Insulin and type I insulin-like growth factor (IGF-I) suppressed growth hormone (GH) expression followed by the induction of prolactin (PRL) biosynthesis in MtT/S cells cultured with normal sera. Insulin also increased the peptidylarginine deiminase activity in a dose-dependent manner. The increase was detectable at 1 ng/ml and reached a maximum (about 16-fold higher than the control) at 1 micrograms/ml. IGF-I showed similar but less prominent effects. The enzyme activity started to increase by 15 hr after the addition of insulin (500 ng/ml), and reached a plateau level at 48 hr. There were concurrent increases in the enzyme mRNA level, enzyme biosynthesis, and enzyme protein contents detected by Northern blot hybridization, [35S]-amino-acid incorporation, and Western immunoblot analysis, respectively. Two-color immunofluorescence staining at 1 day after the insulin addition detected a small number of peptidylarginine-deiminase-positive cells (about 1% of the total cells) which were also GH-positive. The enzyme-positive cells increased to 12% on day 2 and to 24-26% on days 4-6. PRL-positive cells first appeared in the enzyme-positive cell population on day 2, and PRL-positive, enzyme-negative cells appeared later. These results suggest that peptidylarginine deiminase expression increases in association with the hormone switching in MtT/S cells. When the cells were cultured in a steroid-depleted medium, insulin failed to increase the enzyme activity. The insulin action could be specifically restored by estrogen, indicating estrogen-insulin synergism in regulation of the enzyme expression.     

Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.

Antibodies (Abs) raised against the estrogen receptor-alpha (ERalpha) were used to investigate the role of ERalpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17beta-estradiol (E(2)) significantly increased PRL release after 3 or 6 min. When ERalpha Abs that bind specifically to ERalpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERalpha hinge region sequence, increased PRL release in a time- and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E(2). Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERalpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E(2)-BSA. Overall, the data indicate that plasma membrane ERalpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERalpha involved in rapid, nongenomic responses to estrogens.     

Progesterone receptor in the rat anterior pituitary: Effect of estrogen priming and adrenalectomy

The present study was done to determine if a progesterone receptor is present in rat pituitary. Cytosol was labeled with ³H-progesterone (3HP) or ³H-RS020 (³HR) and subjected to sucrose-glycerol density-gradient centrifugation. Serum progesterone was measured for correlation with progesterone receptor levels. Two ³HP-binding peaks (4S + 6S) were evident in uterine and pituitary cytosols. The 4S peak was eliminated by competition with unlabeled cortisol leaving a single 6S peak (progesterone receptor). Estradiol (E) priming of the male or female rat increased progesterone receptor levels in pituitary cytosol as demonstrated using ³HP and ³HR, and pituitary progesterone receptor bound ³HR with a higher affinity than ³HP. Following adrenalectomy of gonadectomized rats, progesterone receptor levels were increased in pituitary and uterine cytosol of both E-primed and unprimed groups. An inverse relationship was established between serum progesterone and progesterone receptor levels in the uterus and pituitary suggesting that stressinduced adrenal progesterone secretion significantly influences progesterone receptor levels in the rat. These results demonstrate an estrogen-inducible progesterone receptor in the rat pituitary with properties similar to those of the uterine progesterone receptor.     

The inhibition of prolactin synthesis in GH3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.     

Immunohistochemical localization of estrogen receptor in rat brain, pituitary and uterus with monoclonal antibodies

Localization of estrogen receptor (ER) in rat brain, pituitary and uterus is shown by the avidin-biotin complex technique using a monoclonal antibody, JS34/32. Immunostaining is observed in nuclei of certain neurons in the preoptic-septal region, hypothalamus and amygdala, and in cells of the anterior pituitary and uterus after estradiol stimulation. Staining is specific since preadsorbed JS34/32 antibody with purified cytoplasmic ER as well as a control monoclonal antibody do not show positive immunoreaction. In the brain, neither cytoplasmic nor nuclear staining is seen in the absence of estradiol stimulation, nor with the progesterone and dihydrotestosterone treatments. The distribution of ER-containing neurons in specific areas of the brain overlaps with the distribution of estrogen target neurons demonstrated by autoradiography. The results demonstrate the usefulness of the monoclonal antibody for the detection of ER in target tissues.     

Cell-specific mechanisms of estrogen receptor in the pituitary gland

Analysis of the mechanism of action of estrogen receptor shows protein and mRNA polymorphism within distinct pituitary receptor-positive cells. The lactotropes exhibit unique properties in these mechanisms that distinguish them from gonadotropes. Therefore, this cell type constitutes an especially interesting model in the male as well as in the female for estrogen receptor studies.Abbreviations PRL prolactin - E2 estradiol - ER estrogen receptor - GnRH gonadotropin releasing hormone - PMSG pugnant mare serum gonadotropin     

Induction of prolactin synthesis in rat pituitary tumor cells by 5-bromodeoxyuridine.

The clonal strain of pituitary tumor cells GH₁2C₁ does not produce detectable amounts of prolactin (<5 ng/mg cell protein per 24 hr), although it does synthesize growth hormone. When GH₁2C₁ cells were grown in the presence of 5-bromodeoxyuridine (BrdU, 3 μg/ml), the cells did produce prolactin as determined by quantitative microcomplement fixation and incorporation of ³H-leucine into ³H-prolactin. BGH₁2C₁ and F₁BGH₁2C₁, two BrdU-resistant (r) substrains derived from GH₁2C₁ which grow in the presence of 30 μg/ml BrdU, also synthesized prolactin (100–500 ng/mg cell protein per 24 hr). Growth of BrdU^r strains was not dependent upon on the presence of the drug in the medium; however, the continued production of prolactin by F₁BGH₁2C₁ cells was dependent upon the presence of BrdU. Growth hormone production in both BrdU^s and BrdU^r strains was not affected by BrdU. Resistance of F₁BGH₁2C₁ cells to BrdU was not due to a defect in BrdU uptake. Thymidine inhibited the incorporation of ³H-BrdU into DNA in both sensitive and resistant strains, and also reduced BrdU-induced prolactin synthesis in F₁BGH₁2C₁. We postulate that induction of prolactin synthesis by BrdU in GH₁2C₁ and F₁BGH₁2C₁ cells is mediated by the incorporation of the drug into cellular DNA. Furthermore, the lack of measurable prolactin synthesis by the parent strain GH₁2C₁ is not due to deletion of the gene for prolactin, but is probably the result of regulatory mechanisms which do not permit expression of this gene.     

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) β subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshβ mRNA levels in all reproductive stages and lower luteinising hormone β (lhβ) mRNA levels in early recrudescence, whereas gh and fshβ mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish. Mol. Reprod. Dev. 79: 592–602, 2012. © 2012 Wiley Periodicals, Inc.