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GROWTH FACTORS-CYTOKINES-ONCOGENES |
Departments of Physiology (K.K., K.Y., W.T., S.T., P.Y., J.A.) and Psychiatry (N.S., S.K.), Yamanashi Medical University, Yamanashi 409-3898, Japan
Address all correspondence and requests for reprints to: Dr. J. Arita, Department of Physiology, Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan. E-mail: . jarita{at}res.yamanashi-med.ac.jp
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Introduction |
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Paradoxically, estrogens also exert antimitogenic actions in the tissues or cell types in which their mitogenic actions are well documented. Estrogen treatment has been reported to induce regression in patients with advanced breast cancer (3) and cause breast cancer of a less advanced clinical stage accompanied by lower mitosis (4). Estrogen treatment also delays and arrests in vivo growth of dimethylbenz[a]anthracene-induced rat mammary carcinoma (5, 6). Mammary tumor cell lines that lack estrogen receptors (ERs) of their own but express exogenous ERs by transfection exhibit a growth-inhibitory response to estrogens (7, 8, 9). In uterine epithelial cells in primary culture, several investigators failed to demonstrate a mitogenic action of estrogens and conversely reported an antimitogenic action (10, 11). The opposing actions of estrogen are also found in the lactotroph, an anterior pituitary cell type that is well known to exhibit a growth stimulatory response to estrogens in vivo and in vitro. Estrogen treatment of rats bearing transplantable MtT/W15 prolactin (PRL)-secreting tumors inhibits tumor cell proliferation (12). Conflicting results have been reported that estrogens stimulate or inhibit in vitro growth and proliferation of cells of the PRL-secreting tumor cell line GH4C1 (13, 14, 15) and human prolactinoma cells (16, 17). Furthermore, discrepancies exist on the effects of estrogens on cell proliferation in other cell types, including ER-negative cells expressing exogenous ERs (18, 19, 20, 21) and cells of colon cancer cell lines (22, 23).
Thus, the antimitogenic action of estrogens has now often been observed in the tissues in which the mitogenic action would be expected, suggesting that signaling pathways involved in mediating these opposing actions exist inherently in the same cell. To date, there is no explanation for these seemingly paradoxical observations, impeding the elucidation and our understanding of the mechanisms underlying estrogen action on cell proliferation. However, the discrepancies may be accounted for to some extent by the idea that estrogens exert opposing actions on cell proliferation in a specific cell type, depending on an as-yet-unidentified cell context. To test this idea, in the present study, we examined whether 17-ß estradiol (E2) has an inhibitory, in addition to a stimulatory, action on the proliferation of rat lactotrophs in primary culture. We characterized the ER-mediated mitogenic and antimitogenic actions of E2. Furthermore, we determined what cell contexts are required for these E2 actions on lactotroph proliferation. We show here that estrogens inhibit cell proliferation only in the presence of growth factors such as insulin and IGF-1.
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Materials and Methods |
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estradiol, progesterone, RU486, 4-hydroxytamoxifen (OHT), forskolin, 3-isobutyl-1-methylxanthine (IBMX), bovine insulin, and human recombinant IGF-1 were purchased from Sigma Chemicals (St. Louis, MO); testosterone was purchased from Wako Pure Chemicals (Osaka, Japan); ICI182,780 (ICI) was purchased from Tocris (Ballwin, MO); and hydroxylflutamide was purchased from Nippon Kayaku (Tokyo, Japan).
Cell culture
Experiments were conducted under the guidelines of the Ethical Committee of Animal Experiments of Yamanashi Medical University. Six-wk-old female Wistar rats purchased from Japan SLC (Shizuoka, Japan) were used to obtain cells for primary cell culture. Anterior pituitary cells were dispersed as described previously (24). Briefly, five anterior pituitaries were minced in MEM for suspension (Sigma) containing NaHCO3, penicillin G, streptomycin, BSA, and HEPES. The mixture was incubated at 37 C in the MEM containing 0.01% trypsin and 0.005% DNase in a siliconized spinner suspension flask with constant stirring for a total of 90 min. The dispersed pituitary cells were treated with trypsin inhibitor and DNase and subjected to cell counting and a viability test. A 100-µl aliquot of cell suspension containing 1.0 x 105 cells in a 1:1 mixture of DMEM and Ham’s nutrient mix F-12 without phenol red and containing 15 mM HEPES, penicillin, and streptomycin (DMEM/F12) (Sigma) was placed in poly-D-lysine-coated culture dishes. The cells were subsequently allowed to attach to the surface of the dishes in a humidified CO2 incubator for 1 h. The pituitary cells were then flooded with 2 ml DMEM/F12 containing 500 ng/ml insulin and precultured at 37 C in a humidified atmosphere of 5% CO2 and 95% air for 1 d.
After the preculture, the pituitary cells were washed with DMEM/F12 and used for experiments. Cultures for experiments were initiated with a serum-free, chemically defined medium (24) containing substances to be tested. The serum-free medium was replaced with fresh serum-free medium every other day during the culture period. For labeling proliferating pituitary cells, cells were incubated with 200 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma) for appropriate times before the end of culture.
At the end of culture, pituitary cells were detached from the culture dishes and redispersed with trypsin as described previously (24). Cells suspended with Earle’s balanced salt solution containing HEPES were attached to poly-D-lysine-coated glass slides by centrifugation with a cytocentrifuge (SC-2, Tomy, Tokyo, Japan). The cells attached on glass slides were fixed with ice-cold methanol and stored in PBS at 4 C until immunostaining for PRL and BrdU.
Immunostaining
The anterior pituitary cells attached to glass slides were double immunostained for BrdU and PRL, as described previously (25). Briefly, the slides were treated with 3 M HCl for 30 min and 10% normal donkey serum (NDS) in PBS for 20 min. Double-labeling immunofluorescence staining was performed in three steps using the following reagents: 1) a mixture of a mouse monoclonal anti-BrdU (Sigma) antibody at a 1:200 dilution and an antirat PRL antibody (NIDDK IC-5) at a 1:4000 dilution; 2) a biotinylated antimouse IgG antibody (Vector Laboratories, Burlingame, CA) at a 1:50 dilution; and 3) a mixture of Texas Red-labeled streptavidin (Amersham Pharmacia Biotech, Arlington Heights, IL) at a 1:50 dilution and a fluorescein isothiocyanate (FITC)-labeled antirabbit IgG antibody (Amersham Pharmacia Biotech) at a 1:50 dilution. The cells were incubated with 100 µl of each reagent diluted with PBS containing 10% NDS for 1 h followed by a 20-min wash.
To immunostain for cyclin D1 in lactotrophs, pituitary cells cultured on dishes were fixed with ice-cold methanol for 30 min. After permeabilization with 0.2% Triton X-100 in PBS for 10 min, cells were washed with PBS, quenched with 3% hydrogen peroxide in PBS for 10 min, and blocked with 10% NDS in PBS for 20 min. For cyclin D1 immunostaining, cells were incubated with a mouse monoclonal antihuman cyclin D1 antibody (DCS-6, NeoMarkers, Union City, CA) at a 1:100 dilution in 10% NDS at 4 C overnight, followed, in order, by an FITC-labeled antimouse IgG antibody (Vector Laboratories) at a 1:100 dilution for 1 h, a horseradish peroxidase-labeled anti-FITC antibody (Roche Molecular Biochemicals, Mannheim, Germany) at a 1:200 dilution for 1 h, and an FITC-labeled tyramide solution (NEN Life Science Products, Boston, MA) at a 1:50 dilution for 20 min. Following the cyclin D1 immunostaining, cells were incubated, in order, with a rabbit antirat PRL antibody at a 1:8000 dilution for 1 h, biotinylated antirabbit IgG antibody (Vector Laboratories) at a 1:100 dilution for 1 h, and Texas Red-labeled streptavidin at a 1:50 dilution for 1 h.
The immunostained slides or dishes were covered with PermaFluor (Immunon, Pittsburgh, PA) and observed with a fluorescence microscope equipped with a dual band mirror unit for FITC and Texas Red.
Statistical analysis
A total of 1000 PRL-immunoreactive cells were examined in randomly chosen fields for each slide to determine the BrdU-labeling index, which was the percentage of pituitary cells immunoreactive for both PRL and BrdU of the total PRL-immunoreactive cells counted. Three slides were analyzed for each treatment group derived from the same cell preparations. Experiments were replicated at least three times with separate batches of cell preparations. Likewise, the number of cyclin D1-immunoreactive cells was counted in a total of 1000 PRL-immunoreactive cells. Differences between groups were statistically analyzed using one- or two-way ANOVA followed by Fisher’s protected least significant difference test.
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, left panel) although, in some experiments in which basal proliferative levels at 48 h were higher, E2 decreased the basal levels. Although basal proliferative levels gradually decreased with culture time in vehicle-treated cells, E2 treatment maintained proliferative levels, leading to a statistically significant 3-fold stimulation at 96 h (P < 0.05). Insulin alone at a dose of 1 µg/ml was highly effective in stimulating basal proliferation throughout the treatment time (Fig. 1, right panel). The insulin-induced proliferation was markedly inhibited by simultaneous treatment with E2 at any time point, although the ratio of proliferative levels induced by a combination of insulin and E2 to those induced by insulin alone was increased with time. After 48 h of E2 treatment, the high levels of insulin-induced proliferation were decreased to 25% of the control levels (P < 0.01). When the inhibitory action of E2 was examined at earlier time points, E2 was found to be significantly effective as early as 12 h (data not shown). Thus, the time points of 88 h and 40 h after E2 treatment were chosen in the following experiments to determine the stimulatory action on basal proliferation and the inhibitory action on insulin-induced proliferation, respectively.
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. A stimulatory action of E2 on basal proliferation was observed at concentrations greater than 1 pM, and a peak level was attained at 100 pM, which was 3-fold higher than the basal level (Fig. 2, left panel). In contrast, an inhibition of insulin-induced proliferation was obtained in the range of 10–1000 pM of E2, with a nadir 20% of the control level (Fig. 2, right panel). Values of the median effective dose for the stimulatory and inhibitory actions of E2 were 8 ± 1 (n = 3) and 85 ± 5 pM (n = 3), respectively, with an 11-fold difference between them.
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). The ratio of proliferative levels induced by a combination of insulin and E2 to those induced by insulin alone was smaller at higher insulin concentrations.
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). Treatment with 17- estradiol, a stereoisomer of E2, failed to inhibit proliferation at 1 nM, but it inhibited it at as much as 10 nM. Unlike these estrogens, neither testosterone and progesterone nor their respective antagonists, hydroxyflutamide and RU486, were active at 1 or 10 nM. These results indicate that the inhibitory action of E2 on insulin-induced proliferation is estrogen specific.
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, treatment with 0.1 nM E2, a maximally effective dose for stimulation, caused a 4-fold increase in basal lactotroph proliferation at 88 h, compared with controls (Fig. 5, left panel), which was suppressed to approximately 5% by simultaneous treatment with ICI or OHT at 10 nM, a 100-fold higher dose than E2 (P < 0.05). An increase in proliferative levels by 1 µg/ml insulin was inhibited to approximately 20% by 1 nM E2, a maximally effective dose for inhibition, at 40 h of cotreatment (Fig. 5, right panel). This inhibition by E2 of insulin-induced proliferation was significantly attenuated by additional treatment with ICI or OHT at 100 nM, a 100-fold higher dose than E2 (P < 0.05), although the antagonistic actions of the antiestrogens were not complete.
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, left upper and lower panels). When their effects on insulin-induced proliferation were determined at 40 h of treatment, both antiestrogens were effective in decreasing insulin-induced high levels of proliferation at 1 nM or more (Fig. 6, right upper and lower panels). Thus, the antiestrogens mimicked the E2 inhibitory action but were less potent than E2, with a maximal inhibition of no more than 50% of controls.
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shows photomicrographs of immunoreactive cyclin D1 in cultured pituitary cells, in which cyclin D1 and PRL are labeled with FITC and Texas Red, respectively. A majority of cultured lactotrophs was negative for cyclin D1, and there was a scattering of lactotrophs with cyclin D1-immunoreactive nuclei (Fig. 7A). When the proportion of these cyclin D1-immunoreactive lactotrophs to total lactotrophs was determined, approximately 2% were found to be immunopositive under basal conditions (Fig. 8). The proportion of cyclin D1-immunoreactive lactotrophs was increased by treatment with 1 µg/ml insulin alone to a 3-fold higher level (Fig. 7B), which was significantly inhibited by simultaneous treatment with 1 nM E2 (Fig. 7C) (P < 0.01).
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). Treatment with IGF-1, an endogenous growth factor in the anterior pituitary gland, at 3 or 30 ng/ml stimulated proliferation in a dose-dependent manner, and the IGF-1-induced proliferation was attenuated by E2, with a pronounced effect in the presence of the higher IGF-1 dose. Serum (5%), a general mitogen used for almost all cultured cells, or combination of forskolin and IBMX (1 µM and 100 µM, respectively), agents known to increase intracellular cAMP concentrations, raised proliferative levels (P < 0.01) with similar mitogenic activities to insulin and IGF-1. Unexpectedly, E2 markedly enhanced rather than attenuated serum- or forskolin/IBMX-induced proliferation (P < 0.01). In contrast, treatment with ICI or OHT alone, which was effective at inhibiting insulin-induced proliferation like E2, had no stimulatory action on serum- or forskolin/IBMX-induced proliferation unlike E2, and ICI rather decreased the proliferation induced by them (P < 0.05) (Fig. 10). Thus, E2 inhibits proliferation not simply in a proliferative activity-dependent manner but in a mitogen-specific manner.
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Discussion |
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These results clearly demonstrate for the first time that estrogens exert opposing actions on cell proliferation even in primary cultures of cells of a normal estrogen-responsive tissue. Although studies using transfection with normal ER into cells of ER-negative breast cancer cell lines have shown that introduction of the ER into ER-negative cells is not sufficient and an additional factor is necessary for restoring estrogen-induced proliferation (7, 8, 9, 18, 20), the model of lactotrophs in primary culture is suggested to contain these components for the expression of both mitogenic and antimitogenic actions of estrogen and express these E2 actions differentially, depending on the cell context. The antimitogenic action of E2 is not proliferative activity dependent; it is mitogen specific. Insulin was used in the present study because it is a potent growth factor that is supplemented in serum-free culture media in numerous studies. However, presumably the mitogenic action of insulin seen in the present study is mediated mostly through the IGF-1 receptor. Of the effective mitogens, IGF-1, a growth factor synthesized within the anterior pituitary gland (29), may play a paracrine or autocrine role in the mitogenic action of E2 on lactotrophs as in the uterus (30) because its production in the anterior pituitary is stimulated by estrogens (31, 32). Similar to our results of the reversal of the mitogenic action of E2 to an antimitogenic action by the cell context, Fujimoto and Katzenellenbogen (33) reported that elevated intracellular levels of cAMP changed antiestrogens from estrogen antagonists to agonists with regard to transcriptional activity.
Importantly, the results of the inhibitory action of E2 on growth factor-induced proliferation of lactotrophs in which E2 stimulates proliferation as well may resolve the discrepancy regarding the effects of estrogens on cell proliferation reported by numerous studies. It is conceivable that the frequent observations of antimitogenic estrogen actions in tumor cells are attributable to the growth factor-dependent nature of the estrogen action. Because many tumor cells in vitro and in vivo secrete a variety of growth factors in an autocrine manner (26) and have enhanced activities of growth factor-mediated signaling pathways to maintain their high proliferative activities (34), the cell context of these tumor cells may allow estrogens to exert an antimitogenic action. Our results would also account for an antimitogenic action of E2 on GH4C1 cells in high cell-density culture (15), in which high proliferation levels might be maintained by autocrine factors. Furthermore, the failure of demonstration of a mitogenic action of estrogens in uterine tissue in primary culture and paradoxical induction of an antimitogenic action may be due to high doses of growth factors in the medium used in those studies (10, 11).
We observed that growth factor-induced lactotroph proliferation was inhibited by estrogens with potencies of E2, estrone, and 17- estradiol in that order, and that the inhibitory action of E2 was antagonized by excess doses of the antiestrogens OHT and ICI. These results suggest that estrogens inhibit the proliferation via ER functions. However, when given alone, the antiestrogens OHT and ICI attenuated insulin-induced proliferation like E2 but less potently. These results are in agreement with those that OHT treatment alone inhibited cell proliferation in other cell types (9, 18, 20). However, ICI also attenuated serum- or forskolin/IBMX-induced proliferation, unlike E2, which inversely enhanced it. Thus, in contrast to the mitogen-specific inhibitory action of E2, the inhibitory actions of the antiestrogens, especially ICI, shown in the absence of E2 were observed in any cell context tested, indicating that they were mitogen independent. The effects of these antiestrogens on the stimulatory and inhibitory actions of E2 on basal and insulin-induced proliferation, respectively, merely reflect their well-known antagonism of E2 actions. However, their inhibitory effects on insulin-, serum-, and forskolin/IBMX-induced proliferation in the absence of E2 are not attributable to their antagonism of E2 action because the chemically defined serum-free culture medium used in the present study contains little E2.
The mechanism by which the antiestrogens inhibit lactotroph proliferation induced by the mitogens other than E2 is unknown at present. The mitogen-independent inhibition by the antiestrogens may be accounted for by the idea that nonestrogenic mitogen-induced proliferation in estrogen-responsive cells requires estrogen-independent ER activity. The idea is based on the findings obtained by numerous studies that blockade of ER functions inhibits growth factor- or cAMP-mediated transcriptional activation and cell proliferation (35). Thus, even though E2 and antiestrogens exert similar inhibitory actions on growth factor-induced lactotroph proliferation, the action of the antiestrogens does not seem to be due to their potential partial agonist activity shown elsewhere (36), and distinct molecular mechanisms may be involved in the antimitogenic actions of E2 and the antiestrogens. Consistent with our results, Zajchowski et al. (37) reported that E2 and OHT required different structural domains of the ER for their antimitogenic activities.
It is currently unknown whether E2 causes the mitogenic and antimitogenic effects on lactotrophs by acting directly on the same lactotroph, or one or both of the two effects are mediated by a distinct anterior cell type. If both of the effects are induced by direct action on a lactotroph, estrogen-occupied ERs could be functionally switched from positive to negative regulators of cell proliferation in the presence of growth factors. However, there is increasing evidence suggesting that estrogens do not directly stimulate cell proliferation in estrogen-responsive tissues and that the estrogen-induced proliferation is caused by growth factors that are secreted from neighboring ER-positive cells in response to estrogens and act in a paracrine manner as a mitogen (26, 30). Using ER-knockout mice to produce uterine tissue recombinants in which epithelium, stroma, or both are devoid of functional ERs, Cooke et al. (38) have clearly demonstrated that E2 stimulates proliferation of the uterine epithelium in tissue recombinants prepared with wild-type uterine stroma but not with ER-knockout uterine stroma. Consistent with these findings, paracrine growth factors such as TGF and -ß and IGF-1 have been implicated in the proliferative response of lactotrophs to E2 (29, 39). Thus, it is possible that the mitogenic action of E2 shown in the present study is not due to a direct action on lactotrophs but is mediated by such a paracrine event. It is quite unknown whether the antimitogenic action of E2 is also mediated by the paracrine event. However, it is possible that, in contrast to the mitogenic action of E2, its antimitogenic action is a direct one on lactotrophs, based on the findings that E2 inhibited uterine epithelial proliferation under culture conditions in which high concentrations of growth factors were present and uterine stromal cells were excluded from culture cell populations (10, 11). It is tempting to speculate that E2 acts on lactotrophs and exerts a direct antimitogenic action so that it prevents specifically the overstimulation of proliferation induced by growth factors that are secreted in a paracrine manner from nonlactotrophs in response to E2 or in an autocrine manner from lactotrophs themselves.
We have demonstrated that inhibition by E2 of insulin-induced proliferation of lactotrophs is accompanied by decreased expression of cyclin D1 protein, which has been postulated to mediate mitogenic actions of both growth factors and estrogens (26, 27), suggesting that modulation of cyclin D1 gene expression plays a role in the antimitogenic action of E2. There are several potential molecular mechanisms linking the antimitogenic action of E2 to a change in gene expression. First, ERs may compete with growth factor-activated transcription factors, leading to transcriptional repression. A transcriptional interference called squelching (40) results from the competition between steroid hormone receptors and other transcription factors for limiting molecules such as coregulators and components of the basal transcriptional machinery. However, it is doubtful that squelching occurs under physiologic conditions or those approximating physiologic conditions, such as the experimental conditions of the present study, which involve only endogenous transcription factors.
Alternatively, the antimitogenic estrogen action may be induced by a direct interaction between ERs and growth factor-activated transcription factors such as AP-1 and nuclear factor 
B (NFB) (41, 42, 43) or a common promoter DNA sequence to which ERs and transcription factors share binding (44). Second, whether estrogens exert either a mitogenic or antimitogenic action on lactotrophs may be determined by the relative expression or activity of coactivators and corepressors in the cell, as proposed for a mixed antagonist/agonist model of tamoxifen (45). This idea is supported by recent findings that agents that stimulate the cAMP- or MAPK-mediated signaling pathway altered levels or activities of the corepressor N-CoR and the coactivator AIB1 that were recruited by and physically interacted with ERs (46, 47, 48). Thus, the experimental condition in which E2 exerts its antimitogenic action may allow ERs to recruit relatively more corepressors available for the E2-mediated inhibition of cell proliferation. Lastly, it seems very likely that ERs act selectively on some level of the signaling pathway of growth factors to inhibit their mitogenic action. The most likely candidate is the MAPK cascade and its upstream molecules because the antimitogenic action of estrogens is accompanied by decreased activity of the MAPK cascade in vascular smooth muscle cells (2). These interactions are supported by recent evidence for a physical association of ERs with molecules involved in the signaling pathway of growth factors such as IGF-1 receptors (49), Src (50), and phosphatidyl inositol-3-kinase (51), although such an association has been implicated in positive, but not negative, functional interactions between ERs and these molecules.
Alternatively, ERs themselves may be target molecules of the interaction between ERs and growth factor signaling pathways. Because numerous studies have shown that the mitogenic action of growth factors requires estrogen-independent ER activity (35), it is tempting to speculate that estrogen binding alters ERs from a conformation that can mediate the mitogenic action of growth factors to another conformation that inversely attenuates the growth factor action with a rapid onset and thereafter allows the mitogenic action of estrogen itself.
To date several interactions have been shown between ERs and growth factors in the regulation of cell proliferation. First, the mitogenic action of growth factors requires ligand-independent activity of ERs (35). Second, the mitogenic action of estrogen requires new synthesis of growth factors that act in an autocrine or paracrine manner (26, 30) and growth factor-mediated signal transduction pathways (19, 21, 52). In addition to these positive interactions, we clearly show here a novel mode of interaction between ERs and growth factors, which is characterized to function negatively. Our results provide new insights into the functional interaction between ER- and growth factor-mediated signaling pathways for cell proliferation. The present study using lactotrophs cultured in serum-free medium has demonstrated that E2 inhibits insulin/IGF-1-induced proliferation but stimulates serum-induced proliferation. The results of the differential effects of E2 on mitogen-induced proliferation suggest the presence in serum of a mitogen(s) that functions opposite to and more potently than insulin or IGF-1. The stimulatory actions of E2 on basal and serum-induced proliferation mimic its well known mitogenic action in vivo, and its antimitogenic action as shown in serum-free culture might be canceled under in vivo conditions by the presence of serum. However, the antimitogenic action is strongly implicated even in in vivo regulation of lactotroph proliferation based on the fact that local concentrations of pituitary-intrinsic growth factors including IGF-1 are much higher than those in serum. The extent to which the antimitogenic action of estrogens is involved in physiologic regulation in vitro and in vivo of estrogen-responsive cell proliferation remains to be determined. Our results that estrogens modulate proliferation of a specific cell by exerting two opposing actions depending on the cell context would also help clarify the mechanism of tissue- and promoter-specific agonist/antagonist reversal as exemplified by selective ER modulators (36).
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E2, 17-ß estradiol; ER, estrogen receptor; FITC, fluorescein isothiocyanate; IBMX, 3-isobutyl-1-methylxanthine; ICI, ICI182,780; NDS, normal donkey serum; OHT, 4-hydroxytamoxifen; PRL, prolactin.
Received December 10, 2001.
Accepted for publication March 26, 2002.
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and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319 and ERß at AP1 sites. Science 277:1508–1510B and C/EBPß. Mol Cell Biol 15:4971–4979[Abstract]
B and estrogen. Mol Endocrinol 15:543–552 rapidly activates the IGF-1 receptor pathway. J Biol Chem 275:18447–18453This article has been cited by other articles:
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  Z. Wang, T. Mitsui, M. Ishida, and J. Arita Adenovirus vectors differentially modulate proliferation of pituitary lactotrophs in primary culture in a mitogen and infection time-dependent manner J. Endocrinol., July 1, 2008; 198(1): 209 - 217. [Abstract] [Full Text] [PDF]
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 M. Ishida, T. Mitsui, K. Yamakawa, N. Sugiyama, W. Takahashi, H. Shimura, T. Endo, T. Kobayashi, and J. Arita Involvement of cAMP response element-binding protein in the regulation of cell proliferation and the prolactin promoter of lactotrophs in primary culture Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1529 - E1537. [Abstract] [Full Text] [PDF]
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 M. Ishida, W. Takahashi, S. Itoh, S. Shimodaira, S. Maeda, and J. Arita Estrogen Actions on Lactotroph Proliferation Are Independent of a Paracrine Interaction with Other Pituitary Cell Types: A Study Using Lactotroph-Enriched Cells Endocrinology, July 1, 2007; 148(7): 3131 - 3139. [Abstract] [Full Text] [PDF]
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M. Candolfi, G. Jaita, V. Zaldivar, S. Zarate, L. Ferrari, D. Pisera, M. G. Castro, and A. Seilicovich Progesterone Antagonizes the Permissive Action of Estradiol on Tumor Necrosis Factor-{alpha}-Induced Apoptosis of Anterior Pituitary Cells Endocrinology, February 1, 2005; 146(2): 736 - 743. [Abstract] [Full Text] [PDF]
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D. Pisera, M. Candolfi, S. Navarra, J. Ferraris, V. Zaldivar, G. Jaita, M. G. Castro, and A. Seilicovich Estrogens sensitize anterior pituitary gland to apoptosis Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E767 - E771. [Abstract] [Full Text] [PDF]
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) or its vehicle (
) in the absence (left panel) or presence (right panel) of 1 µg/ml insulin for varying times and were labeled with BrdU for 18 or 3 h, respectively, before the end of treatment. The BrdU-labeling index of lactotrophs is expressed relative to the group treated with vehicle for 48 h. Data are the mean ± SEM of triplicate determinations from a representative experiment.
), 1 (
), or 10 nM (
