Direct Up-regulation of Estrogen Receptor by Triiodothyronine in Rat Pituitary Tumor MtT/F84

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1 Endocrinol japon 1991, 38 (4), Direct Up-regulation of Estrogen Receptor by Triiodothyronine in Rat Pituitary Tumor MtT/F84 NARIAKI FUJIMOTO, HIROMITSU WATANABE AND AKIHIRO ITO Department of Cancer Research, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima 734, Japan Abstract. To investigate a possible effect of triiodothyronine (T3) on the regulation of estrogen receptor, estrogen dependent rat pituitary tumor, MtT/F84, was studied in rats which received surgical thyroidectomy (Tx) or were given propylthiouracil (PTU) and were supplemented with T3. In T3 loaded rats, the grafted tumor showed high estrogen receptor levels ( % of control), whereas low estrogen receptor levels (20-35% of control) were observed in the tumors grown in Tx and PTU treated rats. A single injection of T3 to Tx rats with MtT/F84 increased the estrogen receptor level in a time dependent manner and reached the maximal level at 6 h. In primary culture of MtT/F84 cells, T3 increased the specific 3H-estrogen binding to tumor cells in a dose dependent manner (165% of control by 10-7M T3) and also in a time dependnet (maximum at 12 h) manner. These data suggest that T3 directly up-regulates the estrogen receptor level in MtT/F84 cells. Key words: Estrogen receptor, Triiodothyronine, Transplantable pituitary tumor, Thyroidectomy. (Endocrinol Japon 38: , 1991) THYROID hormones are essential for normal growth and development in almost all organs [1]. In the pituitary, thyroid hormones have been proven to affect cell proliferation and hormone production in vivo and in vitro [2-6]. Thyroxine is effective in increasing the estrogen receptor level in the pituitary [7, 8]. Synthesis and secretion of PRL are also positively controlled by thyroid hormones [5, 6], although it is under the direct control by estrogen [9]. In rat uterus, thyroid hormones promote the increase in uterine wet weight after estrogen injection [10]. Thyroid hormones seem to modulate estrogenic actions, but the relationship is still unclear. A rat transplantable pituitary tumor, MtT/F84, has estrogen receptor and grows well depending on the dose of estrogen administered [11].It is assumed that the estrogen responsive growth occurs through the receptor [12, 13]. Growth inhibition by estrogen antagonists is evidence that growth is mediated through estrogen receptor. Our previous study has shown that the growth of MtT/F84 is retarded in thyroidectomized rats associated with a decrease in tumor estrogen receptor levels [ 14]. In the present study we examined the changes in the tumor estrogen receptor levels in estrogenized and thyroidectomized rats given T3 and observed the time sequence of estrogen receptor levels. A study was also conducted on the direct action of T3 on the regulation of estrogen receptor in primary cultured MtT/F84 cells. Received: March 15, 1991 Accepted: July 19, 1991 Correspondence to: Dr. Akihiro ITO, Department of Cancer Research, Research Institute of Nuclear Medicine and Biology, Hiroshima University, Kasumi, Minami-ku, Hiroshima 734, Japan. Materials and Methods Animal experiments Female F344 rats, 4-6 weeks of age (purchased

2 FUJIMOTO et al. from Charles River Japan Co., Kanagawa) were used in the following experiments. For the dose response study, rats were divided into 8 groups, each with 6 animals. MtT/F84 tumor tissue was dispersed to cells enzymatically. They were inoculated into all animals at the rate of 4.1 }104 cells/site (2 sites in fat pad subcutaneously/rat). Details of the transplantation have been described previously [11]. All animals also received cholesterol pellets containing 0.5 mg of estradiol subcutaneously on the back at the time of transplantation. Thyroidectomy (Tx) was performed one week before the transplantation. Administration of propylthiouracil (PTU, Sigma Chemicals P-3755) as a 0.05% solution in drinking water was cells/dish) or 100 mm dishes (1 ~107 cells/dish). The medium consisted of Dulbecco's modified Eagle's medium plus Ham's nutrient mixture F12 (DMEM/F12) medium (Sigma Chemicals D8900), penicillin, streptomycin and Dextran coated charcoal (DCC) treated serum (2.5% FCS plus 10% horse serum, Gibco). T3 was dissolved in ethanol, sterilized with a 0.2ƒÊm filter and diluted in medium (<0.1% ethanol as the final concentration). Two days after plating, T3 was added and incubated for given periods (48 h for the dose response study and 0, 3, 6, 12, and 24 h for the time course study). Cells were rinsed with cold PBS (ph 7.4), detached by gentle pipettings and served for binding assay. started one week before the transplantation. Administration of T3 (Sigma Chemicals T-2752) in drinking water at low and high doses of 1.5 ƒêg/l (T3L) and 300ƒÊg/l (T3H) respectively, was begun at the time of transplantation. The grafted sites of tumor cells were observed twice a week. After the tumor grew to 1-2 cm in diameter, the animals were sacrificed under ether anesthesia. Blood samples were collected from the abdominal artery and the sera were stored at -20 Ž until assay. The tumor tissues were frozen with liquid nitrogen and Estrogen receptor assay for tumor tissues Estrogen receptor binding analysis of the cytosol and nuclear extracts of tumor tissue was done as described previously [11, 14]. Briefly, the tumor tissues were homogenized in TED buffer (10 mm Tris, 10 mm EDTA, 1 mm dithiothreitol, ph 7.4) and centrifuged at 800 ~g for 10 min to separate the nuclear pellets. The supernatants were further centrifuged at 105,000 ~g for 50 min to obtain the cytosol. The nuclear pellets were resuspened stored at -70 Ž. in TED buffer containing 0.5 M KC1. After For the time course study in vivo, rats were thyroidectomized, transplanted with dispersed MtT/F84 cells and received cholesterol pellets containing 2.5 mg of estradiol at the time of cell grafts. After the grafted tumor grew to about 1 cm in diameter, the rats were divided into 5 groups of 6 animals. A 2mg/ml T3 solution in saline (containing 10% ethanol) was injected i.p. into all the animals at a dose of 1mg/ 100 g body weight. The rats were sacrificed 3, 6, 12 and 24 h after the T3 injection and cytosolic and nuclear estrogen receptors were measured. incubation in ice both for 60 min, they were centrifuged at 105,000 ~g for 50 min to obtain the nuclear salt extract. Aliquots of the cytosol and nuclear extract were incubated in 2, 4, 6, 7-[3H] estradiol (New England Nuclear) in the range nm with or without excess unlabeled estradiol (5ƒÊM). After incubation at 30 Ž for 40 min, unbounded estradiol was absorbed by the dextran coated charcoal method. The number of maximum binding sites and dissociation constant (Kd) were estimated by Scatchard analysis. Protein concentrations of cytosol were measured by the Lowry method. The Primary culture of MtT/F84 tumor cells MtT/F84 tumors grown in animals receiving 0.5 DNA concentrations were determined by a dye method with bisbenzimidazole (Hoechst 33258). mg of estradiol were collected into MEM under ether anesthesia. The tumor tissues were rinsed, washed twice with MEM and shaken at 37 Ž for 1 h in MEM containing 2 mg/ml collagenase (Wako Chemicals, Tokyo) and 2 mg/ml BSA (Wako Chemicals). The suspension of dispersed cells was passed through nylon mesh and washed twice with MEM. The cells were suspended in culture medium and plated into 60 mm dishes (2 ~106 Specific estrogen binding to whole cells Specific [3H]estradiol binding on the primary cultured cells was measured directly in intact cells. The collected cells were washed twice with PBS. A single concentration of [3H]estradiol (5 nm) with or without 100-fold excess of unlabeled estradiol (0.5ƒÊM) was added to the cell suspension for routine assay. Cells were incubated at 37 Ž for 40

3 REGULATION OF ESTROGEN RECEPTOR BY T1 min and washed three times with ice-cold PBS to remove free estradiol. The cell pellets were dissolved in scintillator containing 33% Triton X-100 and the radioactivity was counted. T3 receptor assay prepared by serial dilutions of T3 (Sigma Chemicals) in 1% DCC treated normal rat serum. The second antibodies, anti-rabbit IgG (for PRL and T3 assays) and anti-monkey IgG (for GH assay), were provided by the Institute of Endocrinology, Gunma University. The method by Oppenheimer, Schwartz, and Surks [1] was followed. Tissues were homogenized in 0.32 M sucrose with 3 mm MgC12 and centrifuged at 700 ~g for 10 min. The pellets were resuspened in 2.4 M sucrose with 3 mm MgC12 and centrifuged at 53,000 ~g for 45 min. The resulting nuclear pellets were resuspended in TMDS buffer (2 mm Tris, 3 mm MgC12, 1 mm dithiothreitol, 0.32 M sucrose, ph 7.4). Aliquots of suspended nuclei were incubated in L-3,5,3'- [125I]T3 (New England Nuclear) at the range of nm with and without 1,000-fold nonradioactive T3 at 30 Ž for 40 min. An equal volume of 2% Triton X-100 was added to terminate the reaction. The nuclei were washed twice with TMDS buffer and the radioactivity was counted. Serum PRL, GH and total T3 levels Radioimmunoassays for PRL and GH were carried out with NIADDK reagents by the recommended methods. Iodination was performed by the lactoperoxidase method with separation on a Sephadex G-75 column. T3 levels were measured by radioimmunoassay with a rabbit antiserum for T3 (MBL, Nagoya) and L-3,5,3'-[1251]T3 (New England Nuclear). The standard solutions were Results Growth of MtTIF84 The results are summarized in Table 1. The appearance of palpable tumors was first observed on day 14 in the control and T3 treated groups. Tumors were detected in T3H treated thyroidectomized rats on day 19 and in T3L treated thyroidectomized rats on day 22. In the PTU treated group, a tumor was first noted on day 26. The growth of MtT/F84 was retarded in hypothyroid animals and T3 treatment resumed it. The tumor growth in hyperthyroid control animals. animals was the same as in Serum T3 levels Total serum T3 levels were measured to confirm the effects of thyroidectomy, PTU and T3 treatments (Fig. 1). Serum T3 levels decreased to 25% of the control level in thyroidectomized animals (p<.05) and 10% in PTU treated animals (p<.01). T3 administration to thyroidectomized rats restored the serum T3 levels. The addition of T3 to normal animals, however, did not increase serum T3 beyond the control level even though the orally administered T3 was the tolerable maximal dose. Table 1. MtT/F84 growth in thyroidectomized (Tx) and/or triiodothyronine treated rats a) Tumor take was evaluated by palpation. Tumors were palpable when the sizes became about 2 mm in diameter. b) Minimum and maximum tumor latency. c) Mean }SEM (in diameter). d) Triiodothyronine was administered in drinking water (T3L: 1.5 ƒêg/l, T3H: 300 ƒêg/l).

4 FUJIMOTO et al. Serum T3 Cytosolic estrogen receptor T3 receptor The T3 receptor was measured in the tumor to investigate whether the T3 receptor is regulated by T3 (Table 2). Only in thyroidectomized rats, the maximum binding sites of T3 receptor were increased significantly. Sequential change in estrogen receptor in vivo After T3 injection, the cytosolic estrogen receptor began to increase at 3 h and reached the maximum value at 6 h (Fig. 2). The nuclear Fig. 1. Serum T3 levels and tumor cytosolic estrogen receptor levels in rats in various thyroid states. *,**: Significantly different from the control value by p<.05 (*) and p<.01 (**). estrogen receptor increased after the increase in cytosolic estrogen receptor and reached the maximum level at 12 h. Serum PRL and GH Estrogen receptor levels In hypothyroid animals, estrogen receptor levels in the tumor were much lower, being 20% of the control value in thyroidectomized rats (p<.01) and 35% in PTU treated rats (p<.01, Fig. 1). The administration of T3 to thyroidectomized rats increased the estrogen receptor levels dose dependently (p<.01). T3H, however, did not restore the estrogen receptor to the control level, although the serum T3 level was the same in both groups. Also in T3H treated normal rats, the estrogen receptor level was 161% of the control. The dissociation constants were similar in the Tx, Tx+T3 and control groups (4.1 }0.78 ~10-10 M in Tx group, 7.2 }1.04 in Tx+T3L, 5.6 }0.25 in the Tx+T3H group and 5.3 }0.78 in the control group, mean }SEM, N=4 or 5). In the PTU and T3 groups, the dissociation constants were significantly higher (9.6 }0.47 ~10-10 M in the PTU group and 9.0 }0.83 in the T3 group). The rats bearing MtT/F84 had high serum PRL and GH levels (Table 3). In the control group, serum PRL and GH levels were 2.8 Đg/ml and 3.0 was almost 1 (0.93). In hypothyroid rats, the GH levels were relatively low. GH/PRL ratios in PTU treated and thyroidectomized rats were 0.07 and 0.19, respectively. Dose response of specific estrogen binding to T3 in primary cultured cells The effects of various concentrations of T3 on specific [3H]estrogen binding are shown in Fig. 3. The specific estrogen binding was increased in good accordance with the T3 concentration range of nm. in the Sequential change in specific estrogen binding in primary cultured cells The change in specific [3H]estrogen binding on the primary cultured cells after adding T3 to the culture medium as the final concentration of 100 Table 2. Triiodothyronine receptor levels in the tumors grown in thyroidectomized and/or triiodothyronine treated rats a) Mean }SEM. b) *: Significantly different from control by p<.05.

5 REGULATION OF ESTROGEN RECEPTOR BY T3 Relative estrogen binding Fig. 3. Dose response increase in specific [3H]estradiol binding caused by T3 in primary cultured MtT/F84 cells. Cells were incubated for 24 h with nm of T3. Individual values are normalized as relative value based on the control value. Each bar indicates the mean }SEM (N=3-5), *,**: Signficantly different from control by p<.05 (*) and p<.01 (**). nm is shown in Fig. 4. The specific estrogen binding increased significantly at 12 h after T3 exposure. Discussion Fig. 2. Sequential changes in cytosolic and nuclear estrogen receptors in MtT/F84 tumor after T3 injection ( œ: after T3 injection, : after injection of saline with 10% ethanol). Each value represents the mean }SEM (N=5-6).*,** : Significantly different from the value at 0 h by p<.05 (*) and p<.01 (**). The present study confirmed our previous ceptor levels in MtT/F84 [14] and demonstrated that the estrogen receptor could be induced by T3 both in vivo and in vitro. MtT/F84 has been characterized also by pituitary hormone secretion. This study showed furthermore that T3 positively regulated GH production, and negatively regulated PRL production in MtT/F84. Table 3. Serum GH and PRL levels in thyroidectomized and/or triiodothyronine treated rats a) Average tumor diameter when rats were sacrificed. b) Mean }SEM. c) *, **: Significantly diferent from control by p<.05 (*) or p<.01 (**). d) Serum GH and PRL levels in intact female F344 rats were 26 }2.7 and 39 }2.9 ng/ml (N=6), respectively.

6 FUJIMOTO et al. Time after 13 addition (hour) Fig. 4. Sequential changes in specific [3H]estradiol binding in MtT/F84 primary cultured cells caused by 100 nm of T3. Each value is normalized as a relative value based on the value at 0 h. Each point shows the mean }SEM (N=3).**: Significantly different from the control value by p<.01. In the pituitary, it has been reported that the estrogen receptor concentration decreased in hypothyroid rats and increased in hyperthyroid rats [7, 8]. In the present study MtT/F84 showed similar changes in estrogen receptor levels both in hypo- and hyperthyroid states. The increase in estrogen receptor corresponded to the serum T3 levels. The supply of T3 to thyroidectomized rat did not restore the estrogen receptor level completely to the control level, whereas the addition of T3 to normal rats increased the receptor to beyond the control level. This suggests that thyroxine or the co-existence of thyroxine with T3 is also important in the physiological regulation of estrogen receptor. The rapid increase in the estrogen receptor concentration after T3 injection in vivo suggests that the increase in the estrogen receptor is directly regulated by T3 and is not a secondary effect due to tumor growth. Furthermore, the increase in the estrogen receptor in the primary cultured cells after the addition of T3 supported the theory of a direct action of T3 in the up-regulation of estrogen receptor. Only a few reports have been appeared on the regulation of estrogen receptor. Leung et al. have reported that prolactin increases the specific incorporation of estrogen into uterine and mammary tissue in vitro [ 15]. In ZR-71-1 human breast cancer cells in vitro, the estrogen receptor is decreased by androgens [ 16]. In MCF-7 cells, auto-regulation of the estrogen receptor has been reported. Estrogen decreased the estrogen receptor levels that were measured by EIA and Northern blot analysis [ 17]. An anti-estrogen such as tamoxifen, on the other hand, increased the estrogen receptor [ 18]. It is not surprising that these estrogen receptor auto-regulating system may exist, although the mechanism is not known. Gardner et al. studied the promoting effects of the uterine response to estrogen by means of thyroid hormones in rats [ 10]. The increase in the uterine wet weight after estradiol injection was greatly diminished in hypothyroid animals, but the estrogen receptor level did not change. Lu et al. investigated the regulation of rat pancreatic glucocorticoid receptors which were decreased by the administration of PTU and increased by thyroxine [19]. The findings seem to be similar to the present results. The inductive effect of T3 on estrogen receptor seems to be similar to the induction of progesterone receptor by estrogen. It is well known that increase in progesterone receptors is promoted by estrogen in the uterus and mammary gland [20, 21]. T3 receptor levels were measured in the tumors grown in various thyroid states in rats. The T3 receptor level in the tumor was nearly the same as in normal rat pituitary [22]. Maximum binding sites of T3 were similar in various thyroid states except in the Tx group. This might indicate an auto-regulation of T3 receptor in this tumor, though the range of change was small. Raaka et al. showed that T3 reduced the amount of T3 receptor in GH1 cells. On the other hand, the affinity of T3 binding was decreased in hyperthyroid rats. This may be caused by the high levels of endogenous T3. Hamada et al. suggested that the values for association constants and maximum binding capacities are falsely low if the amount of endogenous hormones is not taken into account [24]. The serum PRL and GH levels were measured as indicators of tumor function. An increase in serum GH levels caused by T3 could be due to the fact that thyroid hormones are the main regulators of GH production in vivo [25] and in vitro [2]. The ratio of serum GH to PRL may be more essential, because the absolute values for these hormone levels varied among the groups, reflecting the tumor sizes. In W/Fu rats bearing MtT/W15, PTU

7 REGULATION OF ESTROGEN RECEPTOR BY T3 treatment decreased the plasma GH to nearly the normal level, but did not influence PRL production [25]. It has been shown that thyroid hormone increased the ratio of somatotrophs to thyrotrophs and the syntheses of GH and PRL in the rat pituitary [26]. It was shown in the present study that thyroid hormones regulate the estrogen receptor level in MtT/F84 cells in vitro as well as in vivo. Recently, receptors for thyroid hormone, retinoic acid and vitamin D have been extensively investigated and regarded as members of the steroid-thyroid hormone receptor superfamily [27]. It is possible that individual steroid hormone receptors are intimately related, though little is known about the regulation of these receptors. In this respect, MtT/F84 is a good model with which to investigate the roles and mechanisms underlying these regulation. Acknowledgments The authors thank Dr. S. Sakai, Tokyo University, for his excellent technical advice and Miss. Y. Sakai and Miss S. Uemukai for their helpful technical assistance. References 1. Oppenheimer JH, Schwartz HL, Surks MI (1974) Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: Liver, kidney, pituitary, heart, brain, spleen and testis. Endocrinology 95: Samuels HH, Shapiro LE (1976) Thyroid hormone stimulates de novo growth hormone synthesis in cultured GH1 cells: Evidence for the accumulation of a rate limiting RNA species in the induction process. Proceedings of the National Academy of Sciences of the USA 73: Astier HS, DeFesi CR, Surks MI (1980) Kinetics of deoxyribonucleic acid synthesis and replication of thyrotrophs and somatotrophs during development of hypothyroidism and L-triiodothyronine treatment of hypothyroid rats. Endocrinology 106: DeFesi CR, Surks MI (1981) 3,5,3'-triiodothyronine effects on the growth rate and cell cycle of cultured GC cells. Endocrinology 108: Franklyn JA, Wood DF, Balfour NJ, Ramsden DB, Docherty K, Chin WW, Scheppard MC (1987) Effect of hypothyroidism and thyroid hormone replacement in vivo on pituitary cytoplasmic concentrations of thyrotrophn-13 and c- subunit messenger ribonucleic acids. Endocrinology 120: Samuels MH, Wierman ME, Wang C, Ridgway EC (1989) The effect of altered thyroid status on pituitary hormone messenger ribonucleic acid concentrations in the rat. Endocrinology 124: Cidlowski JA, Muldoon TG (1975) Modulation by thyroid hormones of cytoplasmic estrogen receptor concentrations in reproductive tissues of the rat. Endocrinology 97: Altschuler LR, Ceppi JA, Ritt MN, Calandra RS, Zaninovich AA (1988) Effects of thyroxine on oestrogen receptor concentrations in anterior pituitary and hypothalamus of hypothyroid rats. Journal of Endocrinology 199: Lieberman, ME, Maurer RA, Gorski J (1978) Estrogen control of prolactin synthesis in vitro. Proceedings of the Natioal Academy of Sciences of the USA 75: Gardner RM, Kirland JL, Ireland JS, Stancel GM (1978) Regulation of the uterine response to estrogen by thyroid hormone. Endocrinology 103: Ito A, Kawashima K, Fujimoto N, Watanabe H, Naito M (1985) Inhibition by 2-bromo-ƒ - ergocriptine and tamoxifen of the growth of an estrogen-dependent transplantable pituitary tumor (MtT/F84) in F344 rats. Cancer Res 45: Edwards DP, Adams DJ, MuGire WL (1982) Estrogen regulation of growth and specific protein synthesis in human breast cancer cells in tissue culture. In: Leavitt WW (ed) Hormones and Cancer. Plenum Press, New York, pp Lieberman ME, Maurer RA, Claude O, Wiklund J, Wertz N, Gorski J (1982) Regulation of pituitary growth and prolactin gene expression by estrogen. In: Leavitt WW (ed) Hormones and Cancer. Plenum Press, New York, pp Fujimoto N, Roy B, Watanabe H, Ito A (1988) Increase of estrogen receptor level by thyroxine in estrogen dependent pituitary tumor (MtT/F84) in rats. Biochemical and Biophysical Research Communications 152: Leung BS, Sasaki GH (1973) Prolactin and progesterone effect on specific estradiol binding in uterine and mammary tissues in vitro. Biochemical and Biophysical Research Communications 55: Poulin RJ, Simard J, Labrie C, Petitclerc L, Dumont M, Lagace L, Labrie F (1989) Downregulation of estrogen receptors by androgenes in

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