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. 2008 Oct 1;150(2):795–802. doi: 10.1210/en.2008-0756

Estrogen Receptor-α Mediates the Epidermal Growth Factor-Stimulated Prolactin Expression and Release in Lactotrophs

Nira Ben-Jonathan 1, Shenglin Chen 1, Joseph A Dunckley 1, Christopher LaPensee 1, Sanjay Kansra 1
PMCID: PMC2646526  PMID: 18832099

Abstract

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. EGF-receptor (EGFR/ErbB1)-activated Erk1/2 has been reported to activate estrogen receptor (ER) in an estrogen (E2)-independent manner. In the pituitary lactotrophs, both EGF and E2 stimulate prolactin (PRL) release, but the nature of interactions between ErbB and ERα signaling is unknown. Our objectives were to 1) characterize EGF-induced PRL release, 2) determine whether this effect requires ERα, and 3) determine the molecular basis for cross talk between ErbB and ERα signaling pathways. Using GH3 cells, a rat lactotroph cell line, we report that EGF stimulates PRL gene expression and release in a dose- and time-dependent manner. EGF caused a rapid and robust activation of Erk1/2 via ErbB1 and induced phosphorylation of S118 on ERα in an Erk1/2-dependent manner. The global antiestrogen ICI 182780 and the ERα-specific antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP), but not the ERβ-specific antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), blocked the EGF-induced PRL release, indicating an ERα requirement. This was further supported by using ERα knockdown by small interfering RNA. Because the antiestrogens did not block EGF-induced Mek-1 or Erk1/2 phosphorylation, ERα is placed downstream from the ErbB1-activated Erk1/2. These results provide the first evidence that ErbB1-induced PRL release is ERα dependent.

Epidermal growth factor-stimulated prolactin release in lactotrophs is dependent upon estrogen receptor α.

Epidermal growth factor (EGF), acting through EGF-receptors (EGFR), is a potent modulator of cell proliferation/differentiation in a wide variety of cell types. The ErbB family of receptor tyrosine kinases (RTK) include EGFR/ErbB1, ErbB2/Her2, ErbB3, and ErbB4. Upon ligand binding, RTKs undergo a conformational change that activates the intrinsic tyrosine kinase activity of the receptor, leading to increased activation of Erk1/2, Akt, and phospholipase Cγ pathways among others (for reviews see Refs. 1 and 2). Although ErbB overexpression/hyperactivation has been the subject of intense investigations in several tumor models, its specific role in regulating anterior pituitary cell function has not been extensively studied. Expression of EGF, TGFα, and EGFR has been detected in both the normal pituitary and pituitary adenomas, suggesting that ErbB signaling could regulate both hormone production as well as cell proliferation/differentiation (3,4,5,6,7,8,9,10). EGF has been reported to stimulate prolactin (PRL) expression and to induce morphological changes in the rat somatolactotroph cell lines GH3 and GH4 (11,12,13), but the signaling mechanisms that mediate EGF-stimulated PRL release are unknown.

In rodent cells, estrogen (E2) stimulates lactotroph proliferation as well as PRL release (14,15). Support for the importance of E2 in lactotroph function comes from clinical observations, including a higher incidence of prolactinomas in women, increased number of lactotrophs during pregnancy, and positive correlation of lactotroph tumor size with E2 receptor (ER) expression (reviewed in Ref. 16). Although both ERα and ERβ are expressed in GH3 cells (17), ERα appears to play a major role in regulating lactotroph proliferation as well as PRL release in the absence of E2 (18). Indeed, PRL levels are markedly suppressed in ERα-knockout mice (19) but are unaltered in ERβ-knockout mice (20).

The co-overexpression/activation of ErbB receptors and ERs in several tumor types has led to a consensus of a cross talk among these receptors. Two models have been proposed: 1) EGF uses the ER to mediate its biological effect, and 2) E2 uses EGF-R to mediate its effects. The proposed signal transduction pathway in model 1 involves EGFR-activated Erk1/2, which phosphorylates ERα on S118 in the ligand-independent transactivation domain (A/B domain) (21,22,23). This leads to increased ER transactivation even in the absence of E2. This model is supported by the observations that the ability of EGF to stimulate uterine cell proliferation is abolished in ERα-knockout animals and that the antiestrogen ICI 182780 (ICI) blocks the proliferative effects of EGF (24,25,26). Model 2 stipulates that ER through a nongenomic signal transduction pathway causes cleavage of membrane-associated growth factors, leading to activation of RTKs (27,28). This model is supported by the observations that the E2-induced stimulation of cell proliferation is blocked by an anti-EGF antibody and that the ability of E2 to stimulate Erk1/2 activation is blocked by inhibitors of EGFR (29,30).

Although bidirectional interactions between E2 and EGF signaling have been characterized in the breast and the uterus, the status of their interactions in the pituitary lactotrophs is unknown. Using GH3 cells we found that in presence of EGF, ability of E2 to stimulate lactotroph proliferation as well as PRL gene expression, is enhanced. This observation lead us to hypothesize that EGF and E2 may share a common pathway in the control of PRL gene expression, and that EGF may use ER to mediate its biological effects. Our objectives were to: a) characterize EGF-induced PRL gene expression and release, b) determine whether this effect requires ERα, and c) determine the molecular cross talk between ErbB and ERα signaling pathways. We are reporting that the ability of EGF to stimulate PRL gene expression and release is mediated by Erk1/2-induced S118 phosphorylation of ERα.

Materials and Methods

Chemicals and reagents

17β-Estradiol was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human EGF was purchased from Promega Corp. (Madison, WI). ICI 182780, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) were from Tocris (Ellisville, MO). Rat PRL (rPRL) was provided by the National Institute of Diabetes and Digestive and Kidney Diseases. ERα was detected using antibody C-1355 raised against the C terminus of ERα. Anti-Erk1/2 antibody and phosphospecific antibodies for Erk1/2 and S118 ERα were purchased from Cell Signaling Technologies (Beverly, MA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Upstate (Lake Placid, NY). All solvents, buffers, and chemicals were of analytical grade and were purchased from Sigma.

Cell culture

GH3 cells (purchased from American Type Culture Collection, Rockville, MD) were maintained in either Ham’s F10 medium (Life Technologies, Inc./Invitrogen, Grand Island, NY) containing 15% heat-inactivated gelding horse serum (ICN Biomediacls, Aurora, OH), 2.5% fetal bovine serum (FBS) (Life Technologies, Inc./Invitrogen) and penicillin/streptomycin or in DMEM/F-12 50/50 mix (Mediatech, Herndon, VA) containing 10% FBS and penicillin/streptomycin. After trypsinization, cells were washed three times in phenol red-free DMEM/F12 (50/50 mix) containing insulin, transferrin, and selenious acid followed by seeding of cells in the same medium in either 24-well plates or 60-mm dishes. Rat Nb2 lymphocytes, obtained from Dr. A. Buckley (University of Cincinnati, Cincinnati, OH), were maintained in Fischer’s leukemia medium containing 10% heat-inactivated horse serum, 10% FBS, 5 μm 2-β-mercaptoethanol, and penicillin/streptomycin.

Nb2 bioassay for PRL

PRL concentrations in conditioned media (CM) were determined by the rat Nb2 lymphocyte bioassay as described (18). Briefly, cell proliferation in response to rPRL or to appropriately diluted CM from GH3 cells was determined by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphonyltetrazolium bromide assay. To rule out the possibility that EGF, RTK inhibitors (AG1478 and AG825), or Mek-1 inhibitor (UO126) interfere with the bioassay, Nb2 cell proliferation in response to rPRL was determined in the presence or absence of these compounds. Figure 1 shows that none of these compounds altered the proliferative response of Nb2 cells to PRL. We have previously showed lack of effect of antiestrogens on this bioassay and verified that Nb2 cell proliferation was exclusively due to PRL, based on the abolishment of the mitogenic effect of CM from GH3 cells in Nb2 cells by anti-rPRL antibodies (18).

Figure 1.

Figure 1

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Validation of the Nb2 bioassay. Nb2 cells were incubated with rPRL (0–300 pg/well) by itself or in the presence of EGF (A), the receptor tyrosine kinases AG1478 and AG825 (B), or the Erk1/2 inhibitor UO126 (C). The proliferation of Nb2 cells was determined by the MTT assay. Data are expressed as OD and are the mean ± sem of three determinations from a single experiment, which is representative of three separate experiments.

PRL/luciferase reporter gene assays

GH3 cells were seeded in 24-well plates, and 24 h later, cells were transfected with 0.8 μg of the 2.5-kb rat PRL pA3 PRL/luciferase plasmid (described in Ref. 31; a kind gift from Dr. A. Gutierrez-Hartman, Denver, CO); using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s instruction. After 18–24 h, cells were washed three times, and medium was replaced with plating medium containing treatments. Cells were lysed and luciferase activity was determined using a commercial luciferase assay kit (Promega, Madison, WI). Fold change in PRL-luciferase activity was calculated after normalization of relative light units to micrograms of cell protein.

Western blotting

After the various treatments, cells were washed twice in ice-cold PBS and lysed, and total protein content was determined by the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). Equal amounts of cell lysates were separated by SDS-PAGE and the proteins transferred onto polyvinylidene difluoride membranes. After incubation in a blocking buffer, membranes were incubated overnight at 4 C with the primary antibodies (ERα, 1:7500; tERK1/2, 1:1000; pERK1/2, 1:1000; pS118 ERα, 1:1000; actin 1:5000; α-tubulin 1:500). After several washes, membranes were incubated with secondary antibodies for 1 h at room temperature. Antibody-bound proteins were detected by enhanced chemiluminescence (Pierce). Density of protein bands was determined by scanning, using the ImageJ 1.38 × software (National Institutes of Health, Bethesda, MD). Fold changes in response to treatment were calculated after normalization.

ERα knockdown

Using Lipofectamine 2000 (Invitrogen), GH3 cells were transfected with 200 nm ERα SMART pool small interfering (siRNA) or a nontargeting siRNA as a control (Dharmacon, Lafayette, CO). After culturing for 48 h in complete medium, cells were incubated for 24 h in DMEM/F12 (50/50) medium containing ITS (insulin, transferrin, selenious acid) premix (BD Biosciences, San Jose, CA). Cells were then washed twice with the above medium and treated with vehicle or 5 ng/ml EGF for 2 d.

Data analysis

Data are expressed as a mean ± sem. Statistical significance was determined using Student’s t test; a value of P < 0.05 was considered significant.

Results

EGF stimulates PRL release in a dose- and time-dependent manner

To characterize the ability of EGF to stimulate PRL release in GH3 cells, cells were treated with increasing concentrations of EGF for 48 h, and the amount of PRL released into CM was determined by the Nb2 bioassay. Figure 2A shows that EGF stimulated PRL release from GH3 cells in dose-dependent manner, with maximal increase achieved from 5–10 ng/ml. Kinetic analysis showed that EGF significantly induced PRL release after 24 h, with a larger response seen after 48 h. We next questioned whether EGF-induced increase in PRL release involved stimulation of PRL gene expression. To address this issue, GH3 cells were transiently transfected with a PRL/luciferase reporter gene as described in Materials and Methods. Our data show that in response to EGF, there is a dose-dependent increase in PRL gene expression (Fig. 2C) and a good dose-dependent correlation between EGF-induced increases in PRL release as well as PRL gene expression.

Figure 2.

Figure 2

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EGF stimulates PRL release in a dose- and time-dependent manner. A, GH3 cells were treated with various doses of EGF for 48 h. The amount of PRL released into the CM was quantified (micrograms PRL per microgram protein) by the Nb2 bioassay, and data are expressed as percentage of control (Cont). Each value is the mean ± sem of three separate experiments. *, Significant difference from control, P < 0.05. B, Cells were treated with 5 ng/ml EGF for the indicated times. The amount of PRL released into the CM was quantified by the Nb2 bioassay and is expressed as micrograms PRL per microgram protein. Each value is the mean ± sem of three separate experiments. *, Significant difference from corresponding day control, P < 0.05. C, GH3 cells, transiently transfected with rPRL pA3 PRL/luciferase plasmid were treated with various doses of EGF for 24 h, and normalized luciferase activity was determined and expressed as percntage of control. Each value is the mean ± sem of four separate experiments. *, Significant difference from control, P < 0.05.

EGF mediates its effect on PRL release through ErbB1 activation

We next investigated which ErbB receptor is involved. Cells were treated for 48 h with EGF (5 ng/ml) in the presence or absence of the ErbB1-specific inhibitor AG1478 (10 μm) or the ErbB2-specific inhibitor AG825 (12.5 μm). Fig. 3A clearly shows that only AG1478 blocked the stimulatory actions of EGF. We then questioned whether ErbB1 exclusively mediates the EGF-stimulated proximal signaling. For that, GH3 cells were stimulated with 5 ng/ml EGF for 5 min, either alone or after a 1-h pretreatment with the two inhibitors. Erk1/2 phosphorylation was determined by Western blotting using a phosphor-specific anti-Erk1/2 antibody. Within 5 min of exposure, EGF caused a robust stimulation of Erk1/2, and this stimulatory effect was blocked by the ErbB1, but not the ErbB2, inhibitor (Fig. 3B). Taken together, these results indicate that the effects of EGF on lactotrophs are mediated through activation of ErbB1.

Figure 3.

Figure 3

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EGF mediates its effect on PRL release through ErbB1 activation. A, GH3 cells were treated for 48 h with 5 ng/ml EGF alone or in the presence of 1 h pretreatment with the ErbB1-specific inhibitor AG1478 (10 μm) or the ErbB2-specific inhibitor AG825 (12.5 μm). The amount of PRL released into the CM was quantified by the Nb2 bioassay and was calculated as micrograms PRL per microgram protein. Data are expressed as percentage of control. Each value is the mean ±sem of three separate experiments. *, Significant difference from control, P < 0.05; **, significant differences from EGF, P < 0.05. B, GH3 cells were treated with 5 ng/ml EGF for 5 min, either alone or in the presence of the inhibitors as in A. Equal amounts of cell lysates were subjected to Western blotting, with phosphorylated Erk1/2 (pErk1/2, top panel) and total Erk1/2 (tErk1/2, bottom panel) detected as described in Materials and Methods. Results shown are from a single experiment that is representative of three independent experiments.

Erk1/2 inhibition blocks the ability of EGF to stimulate PRL release as well as phosphorylate ERα

Given the robust Erk1/2 activation by ErbB1, we questioned whether such activation is required for EGF to stimulate PRL release. To this end, cells were treated with EGF (5 ng/ml) by itself or were pretreated for 1 h with 10 μm UO126, a Mek1 inhibitor. As evident in Fig. 4A, the ability of EGF to stimulate PRL release was blocked by UO126. In addition, treatment with UO126 alone also caused a significant decrease in PRL release. Subsequently, we examined whether activated ErbB1 phosphorylates ERα in an Erk1/2-dependent manner. As shown in Fig. 4B, EGF caused robust phosphorylation of Erk1/2 as well as ERα within 5 min, and both effects were blocked by UO126, indicating their dependence on Mek-1 activation.

Figure 4.

Figure 4

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ERK1/2 inhibition blocks the ability of EGF to stimulate PRL release as well as to phosphorylate ERα. A, GH3 cells were treated for 48 h with 5 ng/ml EGF alone or in the presence of 1 h pretreatment with the Mek1-specific inhibitor UO126 (10 μm). The amount of PRL released into the CM was quantified by the Nb2 bioassay and was calculated as micrograms PRL per microgram protein. Data are expressed as percentage of control. Each value is the mean ± sem of three separate experiments. *, Significant difference from control, P < 0.05; **, significant differences from EGF, P < 0.05. B, GH3 cells were treated with 5 ng/ml EGF for 5 min, either alone or in the presence of UO126 as in A. Equal amounts of cell lysates were subjected to Western blotting and phosphorylated Erk1/2 (pErk1/2, top panel), total Erk1/2 (tErk1/2, middle panel), and phosphorylated ERα (pS118 ERα, bottom panel) were detected as described in Materials and Methods. Results shown are from a single experiment that is representative of three independent experiments.

ERα mediates EGF-stimulated PRL release

The above results suggested that the two events, ERα phosphorylation and PRL release, might be linked and that ERα mediates the biological effects of EGF in lactotrophs. To address the issue of ERα involvement, we used three types of antiestrogens: ICI, a potent global inhibitor of ERs; MPP, a specific antagonist of ERα; and PHTPP, a specific antagonist of ERβ. As shown in Fig. 5A, addition of ICI caused a dose-dependent inhibition of EGF-stimulated PRL release, with an almost complete blockade at 1–10 nm. Confirmation that ERα rather than ERβ mediates the stimulatory effects of EGF on PRL release comes from the selective effect of the ER-subtype-specific antagonists (Fig. 5, B and C). Notably, both ICI and MPP also caused an EGF-independent suppression of PRL release, consistent with our previous studies that showed E2-independent effects of ERα on the lactotrophs (18). We next questioned whether the antiestrogens would block EGF-stimulated PRL gene expression. To address this issue, GH3 cells were transiently transfected with a PRL/luciferase reporter gene as described in Materials and Methods and were stimulated with vehicle or EGF (5 ng/ml) either by itself or in the presence of ICI (100 nm) for 24 h. Fold change in the normalized luciferase activity shows that the ability of EGF to stimulate PRL gene expression is completely suppressed in the presence of ICI (Fig. 5D).

Figure 5.

Figure 5

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Antiestrogens block the ability of EGF to stimulate PRL secretion. A, GH3 cells were treated for 48 h with 5 ng/ml EGF alone, ICI 182780 alone, or a combination of EGF and ICI. *, Significant difference from control; **, significant differences from EGF, P < 0.05. B, GH3 cells were treated for 48 h with 5 ng/ml EGF (E) alone, the ERα-specific inhibitor MPP (100 nm) alone (M), or a combination of EGF and MPP (M+EGF). *, Significant difference from control; **, significant differences from EGF, P < 0.05. C, GH3 cells were treated for 48 h with 5 ng/ml EGF alone, the ERβ-specific inhibitor PHTPP (100 nm) alone (P), or a combination of EGF and PHTPP (P+EGF). *, Significant difference from control, P < 0.05. In A–C, the amount of PRL released into the CM was quantified by the Nb2 bioassay and was calculated as micrograms PRL per microgram protein. Data are expressed as percentage of control. Each value is the mean ± sem of two to five separate experiments. *, Significant difference from control, P < 0.05; **, significant differences from EGF, P < 0.05. D, GH3 cells, transiently transfected with rPRL pA3 PRL/luciferase plasmid, were treated with vehicle, EGF (5 ng/ml), ICI (100 nm), or a combination of EGF and ICI for 24 h, and normalized luciferase activity was determined in triplicate and expressed as fold change. Each value is the mean ± sem of four separate experiments. *, Significant difference from control; **, significant differences from EGF, P < 0.05.

To further confirm ERα involvement, we used siRNA-mediated ERα knockdown. Compared with the nontargeting control siRNA (Con Si), transfection of GH3 cells with ERα targeting siRNA (ERα Si) caused a 50% decrease in ERα expression (Fig. 6, A and B). We next compared the ability of EGF to stimulate PRL release in ERα-knockdown cells. Fig. 6C clearly demonstrates that EGF stimulated PRL release in control siRNA cells, but its ability to increase PRL release was significantly blocked in the ERα siRNA-treated cells.

Figure 6.

Figure 6

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ERα mediates EGF-stimulated PRL release. GH3 cells were transfected with either nontargeting siRNA (Con Si) or with ERα-targeting siRNA (ERα Si) as described in Materials and Methods. Panel A, After transfections, cells were treated with 5 ng/ml EGF for 2 d. Equal amounts of cell lysates were subjected to Western blotting with an anti-ERα antibody (top panel), followed by stripping and reprobing with anti-actin antibody (bottom panel). Results shown are from a single experiment, representative of four separate experiments. Panel B, After normalization to α-tubulin or actin, data from the Western blots were quantified as described in Materials and Methods, and the percent change from control (Cont) was determined. Data are the mean ± sem of four independent experiments. *, Significant differences from control values, P < 0.05. Panel C, CM from vehicle (C) or EGF-treated Con Si or ERα Si cells were analyzed for PRL by the Nb2 bioassay, data were calculated as micrograms PRL per microgram protein and are expressed as percentage of control (Con Si, vehicle-treated cells). Each value is the mean ± sem of three independent experiments. *, Significant difference from control; **, significant differences from EGF, P < 0.05.

Antiestrogens do not block the ability of EGF to activate ERK1/2

Because the EGF-stimulated PRL release was blocked by the antiestrogens, we questioned whether they act in a nonspecific manner to inhibit ErbB1-mediated signaling. To address this issue, GH3 cells were treated for 5 min with EGF alone or after a 1-h pretreatment with ICI (10 nm) or MPP (100 nm). Western blotting with an anti-phospho-Mek1 antibody, anti-phospho-Erk1/2 antibody, and anti-Erk1/2 antibody revealed that the EGF-induced phosphorylation of Mek1 and Erk1/2 was not affected by the antiestrogens (Fig. 7), indicating that they do not block proximal signaling events of activated ErbB1.

Figure 7.

Figure 7

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The antiestrogens do not block the ability of EGF to activate ERK1/2. GH3 cells were treated with 5 ng/ml EGF for 5 min either alone or in the presence of 1 h pretreatment with the antiestrogen ICI 182780 (10 nm) or the ERα-specific inhibitor MPP (100 nm). Equal amounts of cell lysates were subjected to Western blotting and phosphorylated Mek1 (pMek1, top panel), phosphorylated Erk1/2 (pErk1/2, middle panel), and total Erk1/2 (tErk1/2, bottom panel) were detected as described in Materials and Methods. Results are from a single experiment that is representative of three independent experiments.

Discussion

Both ER and ErbB1 activations have been previously reported to regulate lactotroph function, but whether the regulation of PRL production/release by ER and ErbB1 involves signal cross talk has not been determined. This study shows for the first time that the ability of EGF to stimulate PRL is dependent upon ERα and involves Erk1/2-mediated phosphorylation of ERα at S118.

Our results show a dose- and time-dependent effect of EGF on the stimulation of PRL release from GH3 cells. These data are in good agreement with previous reports (11,12,13). Furthermore, we also established that the EGF-induced PRL gene expression, and therefore the EGF-induced increase in PRL release is not a result of modulation of secretion. The biological effect of EGF on target tissues is mediated by homodimerization of ErbB1 or via heterodimerization with other ErbB family members, with ErbB2 being the favored dimerization partner (32,33,34,35). ErbB2 enhances the ligand affinity as well as ligand-induced phosphorylation of ErbB receptors, prevents receptor internalization, and prolongs signal duration (36,37,38,39,40).

Given the importance of ErbB2 in ErbB1 signaling, together with the observations that ErbB2 is expressed in both normal and tumorous pituitary tissue (41,42), it was important to determine whether the observed effects of EGF are mediated by ErbB2. Our data (Fig. 3) show that the ability of EGF to both activate Erk1/2 as well as to stimulate PRL release from GH3 cells was abrogated only by the ErbB1-specific inhibitor AG1478 and not the ErbB2 inhibitor AG825, confirming the involvement of ErbB1. By comparing the effect of rPRL-induced proliferation of Nb2 cells in the presence or absence of these inhibitors, we ruled out the possibility of their interference with the bioassay (Fig. 1).

Erk1/2 is a classical downstream mediator of activated ErbB1. Our data showed that UO126 not only blocked the ability of EGF to stimulate PRL release but also had an independent inhibitory effect on PRL release, consistent with the prominent role of Erk1/2 in the regulation of PRL expression in lactotrophs (14,43). By using a PRL/Luc reporter gene assay (as in Fig. 2C), as well as Western blotting, we confirmed the inhibitory effect of UO126 in EGF-stimulated PRL gene expression and intracellular PRL levels (data not shown). Furthermore, because ERα is a downstream target of activated Erk1/2, we questioned whether ErbB1-activated Erk1/2 phosphorylates ERα on S118. Our results clearly demonstrate robust phosphorylation of ERα by EGF and its blockade by UO126. These results indicate that in lactotrophs, activated ErbB1 uses ERα in an Erk1/2-dependent manner to mediate its stimulatory effect on PRL release.

If indeed ERα is required for EGF to induce PRL release, then the pharmacological blockade of ERα should prevent EGF from increasing PRL release. This assumption was strongly supported by showing that ICI completely antagonized the ability of EGF to stimulate PRL gene expression and release from GH3 cells (Fig. 5, A and D). This was not due to a nonspecific effect of ICI on ErbB1 signaling, because the ability of EGF to phosphorylate Mek1 as well as Erk1/2 was unaffected by ICI. These results indicate that antiestrogens do not block proximal ErbB1-mediated signaling and also suggest that the interaction between activated ErbB1 and ER occurs downstream of Erk1/2.

Because GH3 cells express both ERα and ERβ, and overexpression of ERβ in these cells results in E2-stimulated PRL release (17), it was important to distinguish between the two ER subtypes. To address this issue, we employed ER-isotype-specific antagonists and showed that only the ERα-specific antagonist MPP completely blocked the EGF-stimulated PRL release. By using a PRL/Luc reporter gene assay (as in Fig. 2C), as well as by Western blotting, we confirmed that MPP blocked EGF-stimulated increase in PRL gene expression and intracellular levels (data not shown). Like ICI, MPP had no effect on the ability of EGF to phosphorylate Mek1 and Erk1/2 (Fig. 7). A further support for ERα involvement came from the demonstration that siRNA-mediated knockdown of ERα abolished the EGF-stimulated PRL release. Together, these results suggest that although ERβ activation is coupled to PRL release under some conditions, i.e. in response to E2, activated ErbB1 signaling by EGF requires ERα, but not ERβ, to stimulate PRL release in lactotrophs.

The expression of ErbB1 and its ligands EGF as well as TGFα are detected in both the normal pituitary and pituitary tumors, suggesting that ErbB1 signaling might contribute to the pathogenesis of prolactinomas (44,45). Using immortalized somatolactotroph cells as well as primary lactotroph cell culture, it has been demonstrated that EGF can modulate lactotroph function (46,47); however, the involvement of ERs in this phenomenon has not been explored. Our study clearly shows that ERα is required for EGF to stimulate PRL production/release. It is of interest to note that TGFα induction by E2 is believed to play a role in E2-stimulated lactotroph proliferation (48). Given our observations that ErbB1-mediated signaling involves ERα, it is reasonable for us to speculate that the E2-induced increases in TGFα levels might be a mechanism for amplifying the E2 responses. Indeed, we have observed (Chen, S., manuscript under preparation) that activated ErbB1 signaling modulates both E2-induced increase in cell proliferation as well as in stimulating PRL gene expression.

Footnotes

This work was supported in part by start-up funds from the Medical College of Wisconsin (S.K.) and the American Cancer Society Pilot Research Grant from the Medical College of Wisconsin Cancer Center (S.K.). Support was also provided by National Institutes of Health Grants ES012212, CA096613, P30-ES06096, and Department of Defense BC050725, and Komen Foundation Grant BCRT87406 (N.B.J.).

Portions of this study were presented at the 88th Annual Meeting of The Endocrine Society, Boston, Massachusetts, June 2006.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 1, 2008

Abbreviations: CM, Conditioned medium; E2, estrogen; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FBS, fetal bovine serum; ICI, ICI 182780; MPP, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1H-pyrazoledihydrochloride; PHTPP, 4-[2-phenyl-5,7-bis (tri-fluoromethyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol; PRL, prolactin; rPRL, rat PRL; RTK, receptor tyrosine kinases; siRNA, small interfering RNA.

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