Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17β (E2) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E2 on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E2, while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.
Prolactin-secreting cells (PRL cells) are generated from preexisting growth hormone-secreting cells (GH cells) in the initial cytogenesis of pituitary cells during the fetal period in mice (Behringer et al., 1988; Borrelli et al., 1989). We have recently demonstrated that PRL cells differentiate from GH cells through mammosomatotrophs (MS cells), which synthesize and secrete both GH and PRL in response to a combined treatment of epidermal growth factor (EGF), insulin and estradiol-17β (E2) in rat pituitary tumor GH3 cells (Kakeya et al., 2000). In contrast, the proliferation of PRL cells is a well-known event in the rat and mouse pituitary gland (Takahashi, 1995). The highest mitotic activity of PRL cells is observed at estrus in the female rat pituitary (Takahashi et al., 1984; Oishi et al., 1993). As estrogen treatment stimulates the proliferation of PRL cells (Lloyd et al., 1975; Takahashi and Kawashima, 1987; Oomizu et al., 2000), the high proliferating activity of PRL cells is caused by the increase in estrogen secretion during the proestrous and estrous days. Thus, it is evident that PRL cells are generated from preexisting PRL cells through mitosis under estrogenic stimulation (self-duplication) in vivo. The above findings indicate that there are two pathways for the cytogenesis of PRL cells: the transdifferentiation of GH cells into PRL cells, and the self-duplication of preexisting PRL cells. However, it remains unclear whether the former pathway operating in GH3 cells requires a mitotic process. The present study attempted to clarify the involvement of mitosis of pituitary cells in the cytogenesis of PRL cells in GH3 cells.
MATERIALS AND METHODS
Two-month-old male rats of the Sprague-Dawley strain obtained from CLEA Japan (Osaka, Japan) were used. Anterior pituitary cells were dispersed by trypsin/DNase digestion following the method of Oomizu et al. (1998). Cell yield was estimated by counting the cells with a haemocytometer. The viability of cells was checked using a trypan blue exclusion test. The dispersed cells were seeded at a density of 3×105 cells/ml in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F12, Sigma Chemical Co., St. Louis, MO) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL, Life Technologies, Inc., Rockville, MD,) on poly-L-lysine (Sigma Chemical Co.)-coated glass coverslips (diameter 13 mm, Matsunami Glass Ind., Osaka, Japan) in a 24-well plate (Becton Dickinson, Lincoln Park, NJ) for 3 days before the hormone treatment. After a 3-day culture, the pituitary cells were maintained in serum-free DME/F12 medium supplemented with hydrocortisone (100 μg/l), 3,5,3′-triiodothyronine (400 ng/l), transferrin (10 mg/l), bovine glucagon (10 ng/l), EGF (10 ng/l), parathyroid hormone (200 ng/l), and sodium selenite (5 μg/l). After the culture under the serum-free condition for 2 days, the pituitary cells were treated with a combination of EGF (10 nM), insulin (300 nM), and E2 (1 nM) for 6 hr or 3 days. These concentrations were determined following the previous reports (Scammell et al., 1986; Kakeya et al., 2000). EGF, insulin and E2 were obtained from Sigma Chemical Co. Experiments were performed under the Guideline of Animal Experimentation, Faculty of Science, Okayama University.
GH3 cells, obtained from the Japan Health Sciences Foundation, Osaka, Japan, were maintained in DME/F12 medium supplementedl with 5% (v/v) FBS at 37°C in a humidified atmosphere of 5% CO2 and 95% air. GH3 cells were cultured in a 24-well plate at a density of 5×104 cells/ml. EGF (10 nM), insulin (300 nM), E2 (1 nM) or a combination of EGF, insulin and E2 were added to the culture medium for 6 days or 9 days. Each group was consisted of three wells, and the medium was changed every 2 days. Independent experiments were performed three times.
Detection of DNA-synthesizing cells
DNA-synthesizing cells were identified by monitoring the uptake of 5-bromo-2′-deoxyuridine (BrdU; Amersham, Buckingham-shire, UK), a thymidine analogue, into the cell nucleus following the previous report (Kakeya et al., 2000). BrdU solution from a cell proliferation kit (Amersham) was added into the culture medium (3 mg/ml) for 3 hr on day 6 or day 9 of the culture. The half of the pituitary cells and GH3 cells treated with BrdU on day 6 were cultured for another 3 days. All cells were rinsed with 0.01M phosphate-buffered saline (PBS; pH 7.4) and fixed in Bouin's solution for 2 hr at room temperature. The cells were dehydrated through a graded series of ethanol concentrations, left in xylene for 5 min, and then rehydrated. Nonspecific immunoreactivity was blocked by incubating the glass coverslips with 1% low-fat dried milk (w/v) in PBS for 30 min at room temperature. The cells were then incubated with monoclonal antibodies raised against BrdU (Amersham) for 1 hr at room temperature in a humidified box. The cells were washed three times with PBS. They were then incubated with peroxidase-conjugated anti-mouse immunoglobulin G (IgG) as a second antibody for 30 min at room temperature. BrdU immunoreactivity was detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.) following the method of Hsu and Soban (1982). After the BrdU detection, the coverslips were incubated in 0.3% H2O2-methanol to inactivate the peroxidase of the second antibodies (peroxidase-conjugated anti-mouse IgG) for the following immunostaining.
Dual-detection of GH and PRL
Dual-detection of GH and PRL in rat pituitary cells was performed with two primary antisera generated from animals of different species. The pituitary cells were first incubated in antiserum against rat PRL (1:4000, NIDDK-anti-rPRL-IC-5, NIH, Bethesda, MD) in a humidified box for 1 hr at room temperature. Detection of PRL was carried out using Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA) and DAB as a chromogen. Subsequent detection of GH was carried out using monkey anti-GH antiserum (1:500, NIDDK-anti-rGH-IC-1, NIH) and tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-monkey immunoglobulin (IgG) (E-Y Laboratories, San Mateo, CA).
Dual immunofluorescent detection of GH and PRL in GH3 cells was performed two primary antisera used for the rat pituitary cells. The cells were first incubated with monkey antiserum against rat GH (1:500, NIDDK-anti-rGH-IC-1) in a humidified box for 1 hr at room temperature, rinsed with PBS three times, and then incubated in rabbit antiserum against rat PRL (1:1000, NIDDK-anti-rPRL-IC-5). The immunostained coverslips were then washed three times with PBS, incubated in TRITC-labeled goat anti-monkey IgG and then fluoresceinisothiocyanate (FITC)-labeled goat anti-rabbit IgG (E-Y Laboratories) in a humidified box for 30 min at room temperature.
Immunofluorescence of each hormone and light field images of BrdU-labeled cells were observed with a laser scanning confocal fluorescent microscope (MRC-600, Bio-Rad Laboratories, Richmond, CA). The fields were scanned across the coverslip to avoid overlap. Each group consisted of three coverslips, and more than 500 cells were observed from each coverslip. The percentage of BrdU-labeled hormone-synthesizing cells was determined by counting the number of those cells against the total cells observed.
The specificity of the antisera for immunocytochemistry was evaluated as follows: (1) incubation of normal rabbit serum instead of each antiserum; (2) preabsorption of each antiserum with corresponding purified antigens (NIDDK rGH I-5 or rPRL I-5, NIH, 50 μg/ml diluted antiserum) 24 hr before the immunostaining. No immunostaining was observed in these controls.
Statistical significance of the difference between the means was assessed with one-way analysis of variance (ANOVA). Three independent experiments were performed in each study. The relative number of GH, MS, and PRL cells was analyzed by the method of R x C test of independence using G-test (Sokal and Rohlf, 1981). Values of the mean and standard errors were calculated from the three independent experiments.
Percentages of GH, PRL, MS and the other cells
The cultured pituitary cell population under the serum-free condition was heterogeneous, consisting of various types of hormone-secreting cells, with GH and PRL cells accounting for approximately one-third of the total pituitary cells (Fig. 1). Treatment with a combination of EGF, insulin, and E2 on pituitary cells induced a slight change in the proportion of GH, PRL, and other cells that secrete neither GH nor PRL relative to the control on day 9 (Fig. 1, p<0.01). During a 3-day culture, the proportions of GH, PRL, and other cells varied in the control and hormone-treatment groups, regardless of treatment (control, p<0.05; the hormone-treated, p<0.01), and the percentages of GH cells declined in both groups.
GH3 cells consist of GH and MS cells, which secrete both GH and PRL, but PRL cells were not detected under the control culture condition (5% FBS supplemented). Treatment with a combination of EGF, insulin, and E2 stimulated the appearance of PRL cells in GH3 cells and altered the proportion of GH, MS, and PRL cells relative to the control on days 6 and 9 of culture (Fig. 2, p<0.001 and p<0.01 respectively). During a 3-day culture, the proportions of GH, MS, PRL, and other cells varied in the control and hormone-treatment groups (p<0.05, in both groups).
DNA-replication in pituitary cells and GH3 cells
DNA-synthesizing pituitary cells, immunocytochemically identified using the BrdU-labeling method, were observed in GH and PRL cells (Fig. 3), indicating that pituitary GH cells and PRL cells have the ability to proliferate under the culture conditions employed. Pituitary cells were treated with BrdU solution for 3 hr on day 6 or day 9 of the culture, and were examined immediately after the treatment. BrdU-labeled cells were immunocytochemically detected (Fig. 4, Group I and III). The other group of pituitary cells that had been treated with BrdU on day 6 was cultured until day 9 (Fig. 4, Group II). BrdU-labeled GH cells and PRL cells were detected regardless of the hormone treatment on days 6 and 9. The additional 3-day culture of GH3 cells after being treated with a combination of EGF, insulin, and E2 did not affect the percentage of BrdU-labeled PRL cells.
Pulse BrdU-labeling for 3 hr was performed as described in the pituitary cells. Fig. 5 shows typical views of BrdU-labeled PRL (#1 cell), GH (#3 cell), and MS cells (#2 cell) in GH3 cells treated with a combination of EGF, insulin, and E2. Fig. 6 illustrates the percentage of BrdU-labeled GH3 cells. BrdU-labeled GH cells and MS cells were detected in both the control and hormone-treated groups, but BrdU-labeled PRL cells were not detected, when BrdU-labeling was performed on days 6 and 9 (Groups I and III). In contrast, when GH3 cells treated with BrdU on day 6 were cultured for another 3 days (Group II), BrdU-labeled PRL cells were detected, and the percentages of BrdU-labeled MS cells and PRL cells increased relative to those of GH3 cells in the control group (p<0.05, p<0.01, respectively), while those of BrdU-labeled GH cells decreased (P<0.05).
Transdifferentiation of GH cells into PRL cells had been previously suggested in several reports (Hoeffler et al., 1985; Frawley and Boockfor, 1991; Takahashi, 1995). Initial differentiation of PRL cells from GH cells is a post-mitotic process in the immature mouse pituitary gland (Behringer et al., 1988; Borrelli et al., 1989). Similarly, we have suggested that the transdifferentiation of GH cells into PRL cells can be induced by treatment of GH3 cells with a combination of EGF, insulin, and E2 (Kakeya et al., 2000). However, it remains unclear whether this appearance of PRL cells is a postmitotic event, as reported in the case of mouse pituitary PRL cells. Therefore, we attempted to clarify the involvement of cell division in the process of PRL cell differentiation observed in GH3 cells. BrdU-labeling was performed to study the DNA-synthesizing cells. The result of the present study demonstrate that PRL cells of GH3 cells cannot be immediately labeled with BrdU, but that BrdU-labeled PRL cells will appear with an additional 3 day-culture. This finding indicates that PRL cells of GH3 cells lose their ability to synthesize DNA and likely their ability to undergo mitosis. Goda et al. (1998) have reported that in the pituitary tumor MtT/S cell line, PRL cells fail to replicate their DNA, which is in agreement with the present study. Despite the inability of the PRL cells to replicate their DNA, BrdU-labeled PRL cells were detected after the additional culture, suggesting that BrdU-labeled GH3 cells other than the PRL cells were trans-differentiated into PRL-only secretors in response to the treatment with a combination of EGF, insulin, and E2.
GH3 cell-populations usually consist of GH and MS cells under the culture conditions employed with the growth of GH3 cells being stimulated (in the medium containing 5% FBS); in contrast, EGF treatment with insulin and/or E2 stimulates the appearance of PRL cells (Kakeya et al., 2000). The appearance of BrdU-labeled PRL cells after the additional 3-day incubation in the present study indicates that PRL cells were generated from preexisting GH cells and/or MS cells that had been labeled with BrdU. Therefore, it is highly probable that the preexisting GH or MS cells directly transdifferentiate into PRL cells without cell division. MS cells are thought to be transitional cells in the process of cytogenesis of PRL cells from GH cells.
Roh et al. (2001) have used a transgenic mouse system targeting the expression of a dominant negative EGF receptor to find that EGF receptor signaling is necessary for the differentiation and/or maintenance of GH and PRL cells in the early cytogenesis of both types of pituitary cells. EGF treatment stimulates PRL gene-transcription and, in contrast, decreases GH gene-transcription in GH3 cells (Kakeya et al., 2000). These findings substantiate the notion that EGF together with insulin and/or E2 is involved in the regulation of PRL cell development. GH gene expression in MS cells may be terminated and PRL gene expression may be started by EGF, and finally, PRL cells (PRL only-secretors) will be generated by treatment with a combination of EGF, insulin, and E2.
It is well known that PRL cells have the ability to replicate DNA and to divide in rat and mouse pituitary glands in vivo and in vitro in response to estrogen and various growth factors (Takahashi et al., 1984; Oishi et al., 1993; Oomizu et al., 2000). In contrast, it is evident from the present study that PRL cells of GH3 cells are unable to replicate DNA molecules. The reason for this discrepancy remains unclear. It is possible, however, that there is a difference in the heterogeneity of cell populations between GH3 and normal pituitary cells, although the major difference between these two cells resides in the cell-immortality. GH3 cells consist of homogeneous cells and are probably derived from a single cell type. In contrast, normal pituitary cells consist of various cell types. Several reports have indicated that an intrapituitary control system, particularly cell-to-cell interactions, is important to the regulation of pituitary function (Schwartz and Cherny, 1992; Denef, 1994; Renner et al., 1996; Vankelecom and Denef, 1997; Schwartz, 2000). Growth factors, which are synthesized in pituitary cells, exert a regulatory function on pituitary cells in a paracrine manner. Therefore, cell division in PRL cells may require various paracrine factors produced in pituitary cells other than PRL cells. Our recent study demonstrated that TGF-α is necessary for estrogen-induced proliferation of pituitary cells (Oomizu et al., 2000). Another interesting finding concerning paracrine factors has been reported by Hentges et al. (2000). They found that basic fibroblast growth factor, produced in folliculo-stellate cells, mediates TGF-β3-induced proliferation of PRL cells. From these findings, it is probable that some factors necessary to DNA-replication and proliferation of PRL cells are deficient in GH3 cells, resulting in the inability of PRL cells to self-duplicate.
Dopamine is well known to inhibit PRL synthesis and release in PRL cells of the normal pituitary glands through its receptors (Caron et al., 1978). GH3 cells do not express dopamine receptors (Cronin et al., 1980). Hence, dopamine fails to inhibit PRL synthesis and release in GH3 cells maintained under normal culture conditions. EGF treatment induces the expression of dopamine receptors in GH3 cells (Missalle et al., 1990), suggesting that dopamine-inhibition of PRL secretion begins to operate. Those previous studies together with the present results indicate that EGF plays a number of roles in the maturation process of PRL cells such as in the stimulation of PRL synthesis, formation of secre-tory granules (Scammell et al., 1986), and induction of dopamine receptors.
Mouihate and Lestage (1995) have reported that estrogen stimulates EGF synthesis in the rat pituitary. EGF and its receptors are expressed in the mouse pituitary (Honda et al., 2000). EGF released from pituitary cells may act on pituitary cells themselves by a paracrine or autocrine mechanism. Zhang et al. (1993) have found that both EGF and E2 stimulate PRL mRNA synthesis and PRL secretion. Judging from these previous reports, it seems possible that estrogen action and EGF action are closely related to the development of MS and PRL cells. As such, the regulatory mechanism concerning EGF production in GH3 cells needs to be studied.
We have recently found in mouse pituitary cells that insulin or IGF-I stimulates the proliferation of PRL cells in vitro, and IGF-I action that is more potent than insulin action (Oomizu et al., 1998). We also demonstrated by in situ hybridization and immunocytochemistry that IGF-I and IGFI receptors are expressed in mouse GH cells, indicating that pituitary IGF-I acts on GH cells in a paracrine or autocrine manner (Honda et al., 1998). Furthermore, GH3 cells express IGF-I protein (Fagin et al., 1987) and IGF-I receptors (Honda et al., 1998). From these findings, it can be assumed that the insulin action shown in the present study may be mediated through IGF-I receptors located on GH3 cells, as insulin at high concentration is known to act on IGF-I receptors (Houslay and Wakelam, 1988).
In conclusion, GH3 cells consist primarily of GH and MS cells in a 5% FBS-supplemented condition. EGF, insulin, and E2 are required for the development of PRL cells. MS cells of GH3 cells are thought to be transitional cells in the process of the transdifferentiation of GH cells into PRL cells. In normal pituitary glands PRL cells proliferate and increase in number in response to estrogen (Takahashi et al., 1984). Thus, there may be at least two pathways for the growth of PRL cells; transdifferentiation of GH cells into PRL cells, and cell division of preexisting PRL cells.
The authors would like to express cordial thanks to Dr. A.F. Parlow and the National Hormone and Pituitary Program, NIDDK, Torrance, CA, USA for supplying antisera. This study was supported in part by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.