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Departments of Physiology (M.I., W.T., J.A.) and Biochemistry (S.M.), Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 409-3898, Japan; and Division of Blood Transfusion (S.I., S.S.), Shinshu University Hospital, Nagano 390-8621, Japan
Address all correspondence and requests for reprints to: Jun Arita, Department of Physiology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 409-3898, Japan. E-mail: jarita{at}yamanashi.ac.jp.
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Introduction |
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Pituitary lactotrophs are typical estrogen-responsive cells. Estrogen treatment in vivo and in vitro induces lactotroph proliferation (7, 8, 9, 10), and its long-term treatment leads to the formation of prolactin (PRL)-secreting tumors (11, 12, 13). There are several proposed mechanisms explaining estrogen action at the pituitary level including structural alterations of the hypophysial portal vasculature (14), decreased responsiveness to the inhibitory hypothalamic hormone dopamine (15), and changes in the synthesis of pituitary-intrinsic growth factors and cytokines (16). In addition to lactotrophs, there are several pituitary cell types that express ERs (17) including folliculostellate (FS) cells. FS cells have multiple functions including scavenging, regulating ion transport, secreting growth factors and cytokines, and promoting angiogenesis in estrogen-induced pituitary tumors (18, 19, 20). Furthermore, it has been proposed that estrogen-induced proliferation of normal lactotrophs is mediated by a cell-to-cell interaction with FS cells (21). In this model, estrogen exerts dual actions to induce lactotroph proliferation: estrogen sensitizes lactotrophs to fully respond to basic fibroblast growth factor (bFGF), and it stimulates TGF-ß3 production in lactotrophs. TGF-ß3 secreted by lactotrophs acts on neighboring FS cells and stimulates the release of bFGF, which, in turn, acts on lactotrophs to induce proliferation (22). The difference between Fischer 344 and Sprague Dawley rats in terms of the susceptibility of their lactotrophs to the mitogenic action of estrogen may be attributable to a strain difference in the paracrine function of FS cells (23). In addition to bFGF, the cytokine IL-6, secreted by FS cells (24), acts on lactotrophs in a paracrine manner to affect lactotroph functions (25, 26), indicating the possibility of the involvement of IL-6 in estrogen-induced lactotroph proliferation.
We have recently shown that estrogen exerts opposing actions on the proliferation of lactotrophs in primary culture, depending upon the cell context (27). Treatment of lactotrophs with 17ß-estradiol (E2) alone or in combination with serum or forskolin, an adenylate cyclase activator, stimulates proliferation. Interestingly, however, E2 inhibits proliferation in combination with insulin or IGF-I. Little is known about the cellular and molecular mechanisms of the antimitogenic action of estrogen on lactotrophs. Therefore, we investigated whether the antimitogenic and mitogenic actions of estrogen require a cell-to-cell interaction between lactotrophs and other pituitary cell types. To do this, we used cell preparations in which heterogeneous anterior pituitary cells were enriched for lactotrophs. In addition to a conventional method of differential sedimentation on a discontinuous Percoll gradient, lactotroph enrichment was performed by fluorescence-activated cell sorting (FACS) of anterior pituitary cells obtained from transgenic rats that express enhanced green fluorescent protein (EGFP) under the control of the PRL promoter. Furthermore, we addressed the putative role of bFGF as a paracrine factor mediating the mitogenic action of estrogen.
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Materials and Methods |
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Cell culture
Anterior pituitaries were obtained from 8-wk-old, female Wistar rats (Crj:Wistar, Charles River, Kanagawa, Japan) at random estrous cycle stages. They were dispersed as described previously (27) and then subjected to Percoll gradient separation or FACS. Lactotroph-enriched and control unenriched cells obtained by these enrichment methods were suspended with a 1:1 mixture of DMEM and Ham’s nutrient mix F-12, without phenol red, containing 15 mM HEPES, penicillin, and streptomycin (DMEM/F12). Cells were plated on poly-D-lysine-coated culture dishes in 100 µl aliquots containing 1.2–2.0 x 105 cells, cultured in a serum-free, chemically defined medium (27), and treated with mitogens for 40 or 88 h. The mitogens used were: E2 (1 nM), IGF-I (30 ng/ml), forskolin (1 µM), and dextran-coated charcoal-treated horse serum (DCC-HS) (10%), which was prepared as described elsewhere (29). In addition, the effect of bFGF was examined using recombinant bovine bFGF (Roche Diagnostics) in combination with rabbit anti-bFGF antibody (R&D Systems, Minneapolis, MN) or normal rabbit IgG (R&D Systems). For labeling proliferating cells, cultured cells were incubated with 200 µM 5-bromo-2'-deoxyuridine (BrdU) (Sigma, St. Louis, MO) for appropriate times before the end of culture. Cells were then redispersed by trypsin and attached to poly-D-lysine-coated glass slides by centrifugation with a cytocentrifuge as described previously (27).
GH3 cells obtained from American Type Culture Collection (Manassas, VA) were grown in Ham’s nutrient mix F-12 medium containing 15% horse serum, 2.5% fetal bovine serum, and antibiotics. Cell suspensions of 1.5 x 106 cells/ml in F-10 were mixed with Ad-Prl/Luc and Ad-CMV/ßgal at multiplicities of infection of 1 and 0.5, respectively, and plated on poly-D-lysine-coated culture dishes in 100-µl aliquots. After 1 h, 2 ml of F-10 containing 10% DCC-HS were added, and cells were cultured for 24 h. GH3 cells were then treated with bFGF in combination with rabbit anti-bFGF antibody or normal rabbit IgG for 48 h.
Differential sedimentation on a discontinuous Percoll gradient
Dispersed pituitary cells were suspended in 0.15 M NaCl, filtered through a 40-µm cell strainer (Becton Dickinson Biosciences, Bedford, MA), and subjected to differential sedimentation on a discontinuous Percoll gradient, as described previously (30) with a minor modification. Pituitary cells were layered on a gradient consisting of 33, 41, and 55% Percoll solutions (Sigma) in 0.15 M NaCl (2 ml of each solution). After the gradient was centrifuged at 560 x g in a swing-out rotor for 20 min at room temperature, cells were recovered as lactotroph-enriched cells from the interface between the top and 33% layers. To obtain control unenriched cells, pituitary cells were mixed with 1 ml of a 33% Percoll solution and left without centrifugation. Cells were washed twice with DMEM/F12 before being used for culture.
Generation of transgenic rats expressing EGFP in lactotrophs
The transgene was constructed on the backbone of the pRL-null vector (Promega) (see Fig. 2A
). A 3.2-kb fragment of the promoter region of the rat PRL gene (position –3221 to +33) isolated from the pSV2A-Prl/Luc plasmid by digestion with BamHI and HindIII was subcloned into pBluescript II SK (+) (Stratagene, La Jolla, CA) at a site created with the same restriction enzymes. A PRL promoter fragment excised with SpeI and SalI from the pBluescript II vector was then inserted upstream of a chimeric intron of pRL-null at a site created with the same restriction enzymes. A 0.8-kb fragment of cDNA encoding EGFP excised by digestion of pEGFP-1 (CLONTECH Laboratories) with BamHI and NotI was subcloned into the pcDNA3.1/Zeo (+) vector (Invitrogen, Carlsbad, CA) at a site created with the same restriction enzymes (pcDNA-EGFP). The luciferase gene of the pRL-null vector was replaced with the EGFP gene of the pcDNA-EGFP plasmid at the NheI and XbaI sites. The entire transgene consisting of the rat PRL promoter, a chimeric intron, the EGFP cDNA, and a Simian virus 40 polyadenylation signal was excised from the pRL-null by SpeI and ApaLI digestion for microinjection (see Fig. 2A).
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). For Southern blot analysis, genomic DNA was digested with EcoRI or PstI, size-fractionated in a 1% agarose gel, capillary blotted onto nylon membrane, and hybridized with a radiolabeled probe of EGFP cDNA excised from pEGFP-1 by digestion with NotI and BamHI. Founders were mated with Crj:Wistar rats to establish transgenic lines that consistently and highly expressed EGFP in the anterior pituitary gland. Among transgenic lines, heterozygous transgenic female rats of the line 11–90 were used for experiments at 8 wk of age. The present study was approved by the Ethical Committee of Animal Experiments of the University of Yamanashi.
Cell sorting
Dispersed anterior pituitary cells obtained from the transgenic line 11–90 rats were filtered through a 40-µm cell strainer and subjected to cell sorting using the FACS Vantage SE (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells were analyzed according to their physical parameters [forward scatter (FSC) and side scatter (SSC)] at a flow rate of less than 2000 counts/sec. Gates 1 and 2 were set on FSC-height/SSC-height and FSC-height/EGFP fluorescence dot plots, respectively, for maximal lactotroph enrichment (see Fig. 2C). Cells that belonged to both gates were sorted as lactotroph-enriched cells, whereas cells distributed within the total range were collected as control unenriched cells. Cells were suspended in DMEM/F12 and used for cultures.
Immunocytochemistry
BrdU-Labeled lactotrophs were detected by double immunostaining for BrdU and PRL as described previously (10). Immunostained glass slides were covered with PermaFluor (Immunon, Pittsburgh, PA) and observed with a fluorescence microscope (Olympus, Tokyo, Japan). A total of at least 1000 PRL-immunoreactive cells were examined in randomly chosen fields for each glass slide, and the BrdU-labeling index was calculated as 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, and experiments were replicated at least three times with separate batches of cell preparations.
To identify the anterior pituitary cell types, dispersed cells were attached to glass slides with centrifugation and fixed with 3% paraformaldehyde (PFA) in phosphate buffer for 10 min. The cells were immunostained by incubation with guinea pig antirat GH (1:600 dilution), LHß (1:1000 dilution), TSHß (1:2000 dilution), or ACTH antibodies (1:1000 dilution) (provided by the National Institute of Diabetes and Digestive and Kidney Diseases and Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA) followed by Alexa-Fluor 594-labeled anti-guinea pig IgG (Molecular Probes, Eugene, OR) at a 1:200 dilution for 1 h. To immunostain for PRL, the cells were incubated with: 1) rabbit antirat PRL antibody (National Institute of Diabetes and Digestive and Kidney Diseases) at a 1:2000 dilution; 2) biotinylated antirabbit IgG (Vector Laboratories, Burlingame, CA) at a 1:200 dilution; and 3) Alexa-Fluor 350-labeled streptavidin (Molecular Probes) at a 1:200 dilution for 1 h. To identify FS cells, cells were stained with: 1) anti-S100 protein antibody (Zymed Laboratories, South San Francisco, CA) at a 1:500 dilution for 1 h; 2) peroxidase-conjugated antirabbit IgG (Jackson ImmunoResearch, West Grove, PA) at a 1:200 dilution for 1 h; and 3) cyanine 5-labeled tyramide solution (Tyramide Signal Amplification, PerkinElmer Life Sciences, Boston, MA) at a 1:50 dilution for 10 min.
For identification of the pituitary cell types that expressed EGFP, dispersed pituitary cells were attached by centrifugation to glass slides that were etched with a grid pattern to permit the relocation of individual cells in various regions of the glass slides. The fluorescence of EGFP in the living pituitary cells was photographed with a digital CCD camera (DP50; Olympus) before PFA fixation. After immunostaining for pituitary hormones, immunostained cells located at the same sites on the grids were photographed again to determine whether they expressed EGFP.
Luciferase assays
The activity of firefly luciferase driven by the rat PRL promoter was assayed using the luciferase assay system (Promega). GH3 cells infected with Ad-Prl/Luc and Ad-CMV/ßgal were lysed by incubation with 400 µl of 1x reporter lysis buffer for 15 min at room temperature followed by vortexing for 15 sec. Cell lysates were centrifuged at 15,000 x g for 5 min. Forty microliters of supernatants were added to 200 µl of the luciferase assay reagent, and the light intensity was measured with a luminometer (BLR-201; Aloka, Tokyo, Japan). The luciferase activity was normalized to ß-galactosidase activity determined using the ß-galactosidase enzyme assay system (Promega) and was expressed in terms of relative luciferase units.
bFGF assays
Cultured pituitary cells treated with vehicle or 1 nM E2 for 88 h were subjected to bFGF determinations. Cultured cells were lysed in 400 µl of fresh culture medium with two consecutive freeze-thaw cycles, and cell lysates were centrifuged at 15,000 x g for 5 min. The bFGF contents in 150 µl of the cell lysate supernatant and 150 µl of culture media collected after the last 48 h culture were determined with a bFGF enzyme immunoassay kit (Quantikine HS; R&D Systems) according to the manufacturer’s protocol. The minimum detectable amount for the bFGF assay as determined by adding 2 SD to the mean OD value of the zero standard was 120 fg/ml.
Statistical analysis
Experiments were replicated at least three times with separate batches of cell preparations. Differences between groups were statistically analyzed using the one-way ANOVA followed by Fisher’s protected least significant difference test.
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). The proliferative response of lactotroph-enriched cells treated for 40 h with E2 in the presence of 10% DCC-HS was comparable with that seen in control unenriched cells (P < 0.01) (Fig. 1B). Treatment with 1 µM forskolin for 40 h increased lactotroph proliferation in both control unenriched and lactotroph-enriched cells, and this response was augmented by E2 in both groups (P < 0.01) (Fig. 1C). We previously demonstrated that 4 d of culture are required for E2 to reliably stimulate proliferation of unenriched lactotrophs in serum-free primary culture (27). Consistent with our previous study, treatment with 1 nM E2 alone for 88 h resulted in stimulation regardless of enrichment (P < 0.01) (Fig. 1D). The E2 dose response curves for the control and enriched cells were similar (data not shown). Because lactotroph enrichment may shorten the latency of 4 d for E2 stimulation, we determined lactotroph proliferation at an earlier time point of E2 treatment. In contrast to the 4 d required for proliferation of unenriched cells, the enriched lactotrophs proliferated after only 40 h of treatment with 1 nM E2 (P < 0.05) (Fig. 1E). These results suggest that the antimitogenic and mitogenic actions of E2 on lactotrophs do not depend on cell-to-cell interactions between lactotrophs and other pituitary cell types.
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The composition of the pituitary cell populations from the line 11–90 rats was determined by immunocytochemistry and compared with that of Wistar rats. There was no difference in the percentage of any cell type between the transgenic and wild-type rats (Table 1). Among the EGFP-expressing cells identified by fluorescence detection in living cells, the percentages of PRL-, GH-, TSHß-, ACTH-, LHß-, and S100-immunoreactive cells were 76.4 ± 9.4, 14.6 ± 0.2, 0.7 ± 0.1, 2.2 ± 0.5, 0.9 ± 0.2, and 3.6 ± 0.8% (n = 3), respectively, indicating that not all of the EGFP-expressing cells were lactotrophs (Fig. 2B). The EGFP-expressing cells were further categorized on the basis of EGFP fluorescence intensities. Cells with high, medium, and low levels of fluorescence represented 7.1 ± 0.3, 24.2 ± 0.8, and 18.0 ± 0.6% of the total anterior pituitary cells, respectively, and contained 98.3 ± 1.0, 74.4 ± 1.7, and 35.0 ± 4.9% PRL-immunoreactive cells, respectively. Because the cells expressing EGFP at a high intensity were most likely lactotrophs, we attempted to selectively collect these cells by setting an appropriate gate for EGFP fluorescence intensity in FACS (Fig. 2C). When cells exhibiting fluorescence intensity levels higher than approximately 100th of the maximal level were sorted, more than 90% of the sorted cell populations were EGFP-expressing cells, as shown by flow-cytometric analysis using cell populations immediately after cell sorting (Fig. 2D). Furthermore, fluorescence microscopic observation after a 2-d culture verified that the cell populations were enriched for lactotrophs by up to 90.3 ± 1.4% (nine experiments) (Table 2). This level of enrichment was greater than that obtained with Percoll gradient separation. The major cell type, other than lactotrophs in the lactotroph-enriched cell population, was the somatotroph. Contamination of the S100-immunoreactive cells or FS cells was decreased from 6.1 to 0.4% as compared with the control unenriched cell population.
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). Likewise, the mitogenic effect of treatment with E2 for 40 h in combination with DCC-HS was observed in both groups (P < 0.01, Fig. 3B). Treatment with forskolin for 40 h stimulated lactotroph cell proliferation in control unenriched (P < 0.01) and lactotroph-enriched cells (P < 0.05). Forskolin-stimulated proliferation was significantly enhanced by simultaneous E2 treatment regardless of enrichment (P < 0.01, Fig. 3C). Treatment with E2 alone for 88 h significantly stimulated lactotroph proliferation in both control unenriched and lactotroph-enriched cells (P < 0.01) (Fig. 3D). We further analyzed the data by expressing the proliferative response to E2 treatment as the fold increase. Unlike in the experiments using Percoll enrichment, enhancement of proliferation of lactotroph-enriched cells was consistently observed in all replicated experiments using FACS enrichment (fold increase in BrdU leveling index in control unenriched cells vs. lactotroph-enriched cells, 6.3 ± 2.2 vs. 13.2 ± 2.4, five experiments, P < 0.05 by the paired t test). Taken together, our results from the FACS-enriched cells confirm the results obtained with Percoll-enriched cells.
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). Next, we determined whether immunoneutralization of endogenous bFGF blocked the mitogenic action of estrogen. To validate the neutralizing potency of the anti-bFGF antibody, we measured its effect on bFGF action in GH3 cells using a parameter of the PRL promoter activity. GH3 cells were infected with Ad-Prl/Luc, an adenovirus vector carrying the PRL promoter-directed luciferase reporter gene and then treated with bFGF alone or in combination with the anti-bFGF antibody. bFGF significantly stimulated the PRL promoter transcriptional activity in a dose-dependent manner in agreement with the results reported by others (31). PRL promoter activities stimulated by 5 ng/ml bFGF were not affected by control normal rabbit IgG but were markedly suppressed after neutralization with 3 or 30 µg/ml of anti-bFGF antibody (P < 0.01) (Fig. 4). In both control unenriched cells and Percoll-enriched lactotrophs, however, neither normal rabbit IgG nor anti-bFGF antibody at any dose was effective in blocking stimulation of lactotroph proliferation induced by treatment with 1 nM E2 for 88 h (P > 0.05) (Fig. 4C). Finally, we determined whether estrogen treatment stimulated production of bFGF as reported elsewhere (23). Using an assay with a sensitivity of 120 fg/ml, bFGF levels were undetectable in the culture media collected from cultures of 2.0 x 105 anterior pituitary cells/dish. bFGF levels remained undetectable, even when cells were cultured at a density of 1.2 x 106 cells/dish (data not shown), or after treatment with 1 nM E2 for 88 h (Fig. 4D, right panel). The intracellular content of bFGF in cells treated with vehicle for 88 h was approximately 3 pg/dish, and this was not altered by E2 treatment (P > 0.05) (Fig. 4D, left panel). Taken together, these results indicate that in our culture conditions, the bFGF-mediated FS cell/lactotroph interaction is not involved in the mitogenic action of estrogen.
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Discussion |
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We successfully enriched anterior pituitary cells for lactotrophs using two different techniques, Percoll gradient separation and FACS. Although contamination of a few FS cells was inevitable in the Percoll-enriched cells, the FACS-enriched population contained negligible FS cells. In these lactotroph-enriched cells, we demonstrated E2-stimulated lactotroph proliferation in three experimental paradigms: treatment with E2 alone for 88 h or E2 treatment in combination with either serum or forskolin for 40 h. Furthermore, we showed that IGF-I-induced lactotroph proliferation was inhibited by E2 in lactotroph-enriched cells to a similar extent as in control unenriched cells. These results suggest that the mitogenic and antimitogenic actions of estrogen do not require a cell-to-cell interaction between lactotrophs and other pituitary cell types. Because enrichment is based on different parameters, the cell density and PRL promoter activity, in Percoll gradient separation and FACS, respectively, the enriched lactotrophs used in this study could have been composed of distinct subpopulations of lactotrophs. Many studies have demonstrated functional differences among lactotroph subpopulations (32, 33, 34). Despite of this, the antimitogenic and mitogenic actions of E2 on lactotroph proliferation were consistently observed in the both enriched cells. This finding supports our conclusion. However, because the lactotroph-enriched cells prepared by Percoll gradient separation and FACS contained about 15 and 10% nonlactotroph cells, respectively, the results of the present study do not completely exclude the involvement of a bFGF-, IL-6- or TGF-
-mediated paracrine interaction between lactotrophs and nonlactotroph pituitary cells as reported by others (21, 26, 35).
The mechanisms by which E2 directly stimulates and inhibits cell proliferation of normal pituitary lactotrophs remain to be determined. One possible mechanism is that estrogen acts on lactotrophs directly to alter cell cycle regulation directly as observed in cell lines and cancer cells (1). Alternatively, estrogen may modify lactotroph proliferation via an autocrine growth factor such as galanin (36, 37), TGF-ß1 (38), TGF- (39), or PRL (40, 41). Notably, in mice carrying a loss-of-function mutation of the endogenous galanin gene, there is almost complete abrogation of the proliferative response of lactotrophs to E2 (37). The estrogen action on proliferation of lactotroph-enriched cells differed from the action on unenriched cells in two ways. First, the fold increase was enhanced in FACS-enriched cells compared with unenriched cells after 88 h of E2 treatment. Second, 40 h E2 treatment alone stimulated lactotroph proliferation in Percoll-enriched cells but not unenriched cells. These observations may be explained by an increase in culture medium levels of an autocrine stimulatory growth factor or a decrease of a paracrine inhibitory growth factor resulting from lactotroph enrichment.
Our study is the first to demonstrate that the antimitogenic action of E2 on a normal estrogen-responsive tissue does not require a cell-to-cell interaction with other cell types. The finding that the mitogenic action of E2 occurred without a cell-to-cell interaction is inconsistent with the paracrine regulation as demonstrated in the uterus and mammary gland (2, 3) but is in agreement with the results of other recent studies (42, 43). The difference between previous studies and ours regarding the involvement of the paracrine cell-to-cell interaction in the mitogenic action of estrogen may reflect tissue-specific mechanisms among the estrogen-responsive tissues. Alternatively, cell-to-cell interactions may be a function of the developmental stages and the dose of steroids administered. For example, the epithelial ER is required for estrogen-induced proliferation of mammary epithelial cells in adult mice (42), but not in immature mice (3), and that the stromal ER is sufficient only in the presence of high doses of estrogen and progestin.
It has been proposed that estrogen stimulates proliferation of normal lactotrophs via a cell-to-cell interaction mediated by a TGF-ß3-bFGF loop between lactotrophs and FS cells (21). Oomizu et al. (23) reported that lactotrophs enriched by Percoll gradient separation and cultured in the presence of 10 nM E2 did not proliferate unless cocultured with FS cells. Additionally, bFGF treatment of lactotroph-enriched cells stimulated lactotroph proliferation in the presence of estrogen (22), and E2 treatment of an FS cell line increased bFGF levels in cell lysates and the culture medium (23). However, the results of our study argue against the bFGF-mediated paracrine interaction. Enrichment using Percoll or FACS did not affect lactotroph proliferation induced by treatment with E2 alone. Additionally, simultaneous treatment with E2 and bFGF inhibited lactotroph proliferation. E2 treatment did not affect cellular bFGF contents. Finally, sufficient immunoneutralization of endogenous bFGF did not block E2-induced proliferation. The discrepancy in the results between their studies and ours may be partly due to a difference in cell culture conditions. Pituitary cells in the present study were obtained from randomly cycling female rats and cultured without serum, whereas pituitary cells in their studies were obtained from ovariectomized, chronically E2-treated rats and were transiently cultured with medium containing serum before estrogen treatment. E2 treatment in vivo is known to profoundly influence pituitary cell functions and changes the secretion of pituitary growth factors. Long-term treatment, in particular, induces estrogen-dependent hyperplasia and tumor formation in estrogen-sensitive strains such as Fischer 344 rats (44). Thus, differences in culture conditions could affect cell-to-cell interactions and subsequent paracrine effects. However, the discrepancy that opposite actions of bFGF on lactotroph proliferation were observed in their study and ours remains to be resolved.
We generated transgenic rats that expressed EGFP under the control of the PRL promoter. Although a high level of EGFP expression was exclusive to the lactotroph, EGFP expression was also observed in nonlactotroph pituitary cells, predominantly somatotrophs. Recognizing this, we restricted our collections to cells with higher EGFP fluorescence intensities, thereby excluding the somatotrophs. Because the expression of EGFP in nonlactotroph cells was observed in not only the line 11–90 but also other transgenic lines that we generated using the same PRL promoter, the aberrant EGFP expression in the transgenic rats is not due to the position effect whereby local enhancers may affect expression. The aberrant expression may be attributable to incomplete specificity of the 3.2-kb PRL promoter used in this study. A rat PRL promoter with a similar length to ours was used to transgenically express TGF- and nerve growth factor in lactotrophs in previous studies (39, 45). Furthermore, immunohistochemical analysis performed by Crenshaw et al. (46) revealed the lactotroph-specificity of a 3-kb PRL promoter using transgenic mice. However, the sensitivity of fluorescence detection of EGFP in enzymatically dispersed, spherical living cells is greater than that of immunohistochemical detection of protein expression in fixed cell-overlapping tissues. Thus, even a low level of PRL promoter-driven protein expression, undetectable in previous studies, may have been detected in our study. Consequently, the 3.2-kb PRL promoter is sufficient for lactotroph-specific protein expression in vitro but may not be appropriate for use with transgenic animals. Another observation in our transgenic rats was that the EGFP-fluorescence and PRL-immunostaining intensities were not necessarily correlated within the same cells. Some lactotrophs exhibited weak EGFP fluorescence and strong PRL immunostaining as shown in Fig. 2B. This difference may be due to differences in intracellular localization and the subsequent protein processing: PRL is stored in secretory vesicles and released extracellularly, whereas EGFP exists in the cytoplasm and is subjected to enzymatic degradation.
In conclusion, estrogen does not require a paracrine cell-to-cell interaction with other pituitary cell types to exert its mitogenic and antimitogenic effects on lactotrophs. Unlike the uterus and mammary gland, pituitary lactotrophs self-regulate their ER-mediated estrogen receptivity and the subsequent proliferative response. An autocrine mechanism mediated by growth factors produced by lactotrophs themselves is likely to be involved in the estrogen action, and further studies are required to elucidate it.
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Acknowledgments |
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Footnotes |
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First Published Online April 5, 2007
Abbreviations: bFGF, Basic fibroblast growth factor; BrdU, 5-bromo-2'-deoxyuridine; DCC-HS, dextran-coated charcoal-treated horse serum; E2, 17ß-estradiol; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; FACS, fluorescence-activated cell sorting; FS, folliculostellate; FSC, forward scatter; PFA, paraformaldehyde; PRL, prolactin; SSC, side scatter.
Received November 8, 2006.
Accepted for publication March 26, 2007.
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References |
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and ß in the rodent mammary gland. Proc Natl Acad Sci USA 97:337–342 and ß in the rat pituitary gland. Endocrinology 139:3976–3983 in the mouse pituitary. Zool Sci 20:83–89[CrossRef][Medline] expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 136:4479–4488[Abstract]. Endocrinology 143:2357–2365 is required for proliferation and morphogenesis in the mammary gland. Proc Natl Acad Sci USA 103:2196–2201This article has been cited by other articles:
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  M. C Bunck, M. Debono, E. J Giltay, A. T Verheijen, M. Diamant, and L. J Gooren Autonomous prolactin secretion in two male-to-female transgender patients using conventional oestrogen dosages BMJ Case Reports, August 10, 2009; 2009(aug10_1): bcr0220091589 - bcr0220091589. [Abstract] [Full Text]
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S. Zarate, G. Jaita, V. Zaldivar, D. B. Radl, G. Eijo, J. Ferraris, D. Pisera, and A. Seilicovich Estrogens exert a rapid apoptotic action in anterior pituitary cells Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E664 - E671. [Abstract] [Full Text] [PDF]
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 L A Nolan and A Levy Prolonged oestrogen treatment does not correlate with a sustained increase in anterior pituitary mitotic index in ovariectomized Wistar rats J. Endocrinol., March 1, 2009; 200(3): 301 - 309. [Abstract] [Full Text] [PDF]
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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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