Estrogen receptor-alpha mediates the epidermal growth factor-stimulated prolactin expression and release in lactotrophs

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 ERalpha signaling is unknown. Our objectives were to 1) characterize EGF-induced PRL release, 2) determine whether this effect requires ERalpha, and 3) determine the molecular basis for cross talk between ErbB and ERalpha 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 ERalpha in an Erk1/2-dependent manner. The global antiestrogen ICI 182780 and the ERalpha-specific antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP), but not the ERbeta-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 ERalpha requirement. This was further supported by using ERalpha knockdown by small interfering RNA. Because the antiestrogens did not block EGF-induced Mek-1 or Erk1/2 phosphorylation, ERalpha is placed downstream from the ErbB1-activated Erk1/2. These results provide the first evidence that ErbB1-induced PRL release is ERalpha dependent.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

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.