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Molecular Endocrinology 20 (12): 3321-3335
Copyright © 2006 by The Endocrine Society

Mechanisms for Growth Factor-Induced Pituitary Tumor Transforming Gene-1 Expression in Pituitary Folliculostellate TtT/GF Cells

G. Vlotides, M. Cruz-Soto, T. Rubinek, T. Eigler, C. J. Auernhammer and S. Melmed

Division of Endocrinology (G.V., M.C.-S., T.R., T.E., S.M.), Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048; and Department of Internal Medicine II (C.J.A.), Klinikum der Ludwig-Maximilians-Universität München, Standort Grosshadern, Munich 81377, Germany

Address all correspondence and requests for reprints to: Dr. Shlomo Melmed, Academic Affairs, Room 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY TUMORS ACCOUNT for 15% of all intracranial neoplasms and are discovered in up to 25% of unselected autopsy specimens (1). Although the clonal composition of pituitary adenomas attests to the cellular basis of pituitary tumorigenesis, their generally benign nature and high level of differentiation suggest an important role of the intrapituitary microenvironment in promoting transformation or proliferation in genetically transformed cells (1, 2). Several hormones, growth factors, and cytokines contribute to this pituitary microenvironment. Hormonally inactive pituitary folliculostellate (FS) cells are particularly important for pituitary cell paracrine regulation, by their ability to respond to, and secrete, a variety of growth factors and cytokines (3, 4, 5, 6).

Epidermal growth factor (EGF) frequently stimulates cancer growth and also regulates normal pituitary hormone secretion and cell proliferation (7). Although the EGF receptor (EGFR) is expressed in pituitary adenomas (8, 9), little is known of EGFR-mediated tumorigenic mechanisms within the pituitary.

Pttg1, a pituitary-derived transforming gene (10), is overexpressed in a variety of tumors, including pituitary (11), thyroid (12), colon (13), and breast tumors (14). In some tumors, high Pttg1 levels correlate with invasiveness (13, 15, 16, 17), and Pttg1 has been identified as a key signature gene associated with tumor metastasis (18). Pttg1 overexpression induces cell transformation in vitro, and tumor formation in nude mice (10, 19, 20) and Pttg1-transfectant-derived conditioned medium induces angiogenesis (21). PTTG1 is a transcriptional activator (22) and stimulates growth factor expression including fibroblast growth factor-2 and vascular endothelial growth factor A (21, 23). A PTTG binding factor (PBF) facilitates PTTG nuclear translocation and potentiates its transcriptional function (24).

Structural homology with yeast securin led to the identification of PTTG1 as a vertebrate securin critical in regulation of sister chromatid separation during mitosis (25). Live imaging of single cells revealed that PTTG1 accumulation, as a consequence of overexpression, inhibits mitosis progression and chromosome segregation but does not directly affect cytokinesis, resulting in aneuploidy (26). Thus, PTTG1 exhibits multifunctional properties, and the underlying contribution to tumorigenic mechanisms is only partially understood.

PTTG1-associated stimulation of growth factor expression and angiogenesis (21, 23) may be important mechanisms for autocrine/paracrine tumorigenic feedback. However, understanding PTTG1 regulation and expression within different pituicyte populations remains limited.

PTTG1 expression in pituitary FS cells (FS cell line TtT/GF) (27) was therefore tested. Agranular FS cells are hormonally inactive and constitute a small proportion of nonfunctioning pituitary adenoma cell types (28) but represent about 5–10% of the anterior pituitary cell mass (29). FS cells are the major intrapituitary source of fibroblast growth factor-2 (30) and vascular endothelial growth factor A (31), which are both induced by PTTG1 (21, 23), and both growth factors are abundant in pituitary tumors (23, 32).

The aim of this study was to examine the effect of EGF on FS PTTG1 expression and cell proliferation and to characterize intracellular pathways involved in eliciting these effects. We report EGFR expression in TtT/GF cells and EGFR-mediated induction of PTTG1 expression associated with increased cell proliferation. EGF-induced Pttg1 expression is cell cycle-dependent and involves protein kinase C (PKC), phosphatidylinositol 3 kinase (PI3K), and MAPK intracellular pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EGF and TGF{alpha} Induce Pttg1 mRNA Expression and Cell Proliferation in TtT/GF Cells
Using RT-PCR, a 319-nucleotide (nt) fragment of murine EGFR cDNA, a 566-nt fragment of murine Pttg1, and a 292-nt fragment of murine PBF were generated in TtT/GF FS cells (Fig. 1Go).


Figure 1
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  		Fig. 1. Expression of Pttg1, PBF, and EGFR mRNA in Murine FS TtT/GF Cells

RT-PCR was performed on three different RNA samples. RT+, With RT; RT-, without RT. A 566-nt fragment of murine Pttg1, a 292-nt fragment of murine PBF, and a 319-nt fragment of the murine EGFR cDNA were generated and verified by sequencing.

 
EGF (5 nM) induced Pttg1 mRNA expression 1.9 ± 0.3-fold at 18 h and 3.4 ± 0.8-fold (P < 0.001) at 24 h (Fig. 2AGo). EGF also enhanced proliferating cell nuclear antigen (PCNA) mRNA expression approximately 2-fold between 18 and 24 h (Fig. 2AGo). PBF mRNA expression, however, was not altered. Increased Pttg1 and PCNA mRNA expression was associated with approximately 3-fold enhanced entry of cells into the S-phase of the cycle between 18 and 24 h (Fig. 2AGo). EGF (up to 50 nM) dose-dependently stimulated Pttg1 expression approximately 2- to 3-fold above baseline (Fig. 2BGo).


Figure 2
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  		Fig. 2. Time and Dose-Dependent Effects of EGF on Pttg1, PBF, and PCNA mRNA Expression and Cell Proliferation

TtT/GF cells were serum-starved for 20 h and subsequently treated with indicated concentrations of EGF. Total RNA was extracted at the times indicated, and Pttg1 mRNA expression determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratio of Pttg1 or PCNA mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio or PCNA/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 and PCNA mRNA expression levels of other groups were normalized to this control group. At the times indicated, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. A, Time-dependent effects of EGF (5 nM). Relative Pttg1 and PCNA mRNA expression (mean ± SEM) of four independently performed experiments (upper panel). **, P < 0.01; ***, P < 0.001. A representative experiment of four independently performed experiments is shown (middle panel). Cell cycle analysis of four independently performed experiments (lower panel). B, Dose-dependent effects of EGF. TtT/GF cells were treated with the indicated EGF concentrations for 20 h. A representative experiment of two independently performed experiments is shown.

 
The EGFR ligand TGF{alpha} (5 nM) induced Pttg1 mRNA expression 1.7 ± 0.3-fold at 18 h and 2.4 ± 0.6-fold (P < 0.05) at 24 h (Fig. 3AGo). TGF{alpha} also increased PCNA mRNA expression approximately 2-fold (Fig. 3AGo). PBF mRNA expression was unaltered. Increased Pttg1 and PCNA mRNA expression was associated with approximately 2.5-fold enhancement of cell entry into the S-phase between 18 and 24 h (Fig. 3AGo). Results of a representative experiment showing TGF{alpha} dose-dependent Pttg1 induction are depicted in Fig. 3BGo.


Figure 3
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  		Fig. 3. Time and Dose-Dependent Effects of TGF{alpha} on Pttg1, PBF, and PCNA mRNA Expression and Cell Proliferation

TtT/GF cells were serum-starved for 20 h and subsequently treated with indicated concentrations of TGF{alpha}. Total RNA was extracted at the time points indicated, and Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratio of Pttg1 mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 mRNA expression levels of other groups were normalized to this control group. At the times indicated, cells were fixed and cell cycle was analysis performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. A, Time-dependent effects of TGF{alpha} (5 nM). Relative Pttg1 mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). **, P < 0.01. A representative experiment of three independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel). B, Dose-dependent effects of TGF{alpha}. TtT/GF cells were treated with the indicated TGF{alpha} concentrations for 20 h. A representative experiment of two independently performed experiments is shown.

 
EGFR Antagonists Suppress EGF-Induced Pttg1 mRNA Expression and Cell Proliferation
Pretreatment with AG1478 (500 nM) for 60 min blocked EGF-induced Pttg1 (P < 0.001) and PCNA (P < 0.01), as well as EGF-induced entry of cells into S-phase (Fig. 4AGo). To confirm the specificity of these observations, TtT/GF cells were pretreated with the tyrosine kinase inhibitor gefitinib for 60 min (Fig. 4BGo). As low as 1 µM gefitinib abolished EGF-induced Pttg1 and PCNA mRNA expression, as well as EGF-induced entry of cells into S-phase (Fig. 4BGo).


Figure 4
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  		Fig. 4. EGFR Antagonists Suppress EGF-Induced Pttg1 mRNA Expression and Cell Proliferation

TtT/GF cells were serum-starved for 20 h and were subsequently treated with AG1478 (500 nM) or gefitinib (1 and 10 µM) for 1 h before stimulation with EGF (5 nM). Total RNA was extracted 20 h after EGF induction. Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratio of Pttg1 mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 mRNA expression levels of other groups were normalized to this control group. Twenty hours after EGF induction, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells by the G2/M-phase of the cell cycle by gray bars. A, Inhibition of EGF-induced Pttg1 and PCNA mRNA expression and cell proliferation by AG1478. Relative Pttg1 mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). **, P < 0.01; ***, P < 0.001. A representative experiment of three independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel). B, Inhibition of EGF induced Pttg1 and PCNA mRNA expression and cell proliferation by gefitinib. Relative Pttg1 mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). **, P < 0.01; ***, P < 0.001. A representative experiment of three independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel).

 
Signaling Blockers Suppress EGF-Induced PTTG1 Expression and Cell Proliferation
Pretreatment of TtT/GF cells with the PI3K inhibitor LY294002 (10 µM; 60 min) and MAPK kinase/ERK kinase (MEK) inhibitor U0126 (10 µM; 60 min) abolished EGF-induced Pttg1 (P < 0.001) and suppressed PCNA induction, as well as EGF-induced entry of cells into S-phase (Fig. 5Go).


Figure 5
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  		Fig. 5. PI3K and MAPK Signaling Blockers Suppress EGF-Induced Pttg1 mRNA Expression and Cell Proliferation

TtT/GF cells were serum-starved for 20 h and were subsequently treated with DMSO (0.1%), U0126 (10 µM), or LY294002 (10 µM) for 1 h before stimulation with EGF (5 nM). Total RNA was extracted 20 h after EGF induction and Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratio of Pttg1 mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 mRNA expression levels of other groups were normalized to this control group. Twenty hours after EGF induction, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. Relative Pttg1 mRNA expression (mean ± SEM) of four independently performed experiments (upper panel). *, P < 0.05; ***, P < 0.001. A representative experiment of four independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel).

 
To examine the effect of classical PKCs (cPKCs) and novel PKCs (nPKCs) on EGF-induced Pttg1 expression, TtT/GF cells were pretreated (60 min) with the competitive diacylglycerol inhibitor calphostin C (3 µM; inhibition of cPKCs and nPKCs), with the selective inhibitor of Ca2+-dependent PKCs Gö6976 (3 µM; inhibition of cPKCs but not nPKCs) and rottlerin (3 µM), the selective nPKC {delta} and {theta} inhibitor. Pretreatment with calphostin C, Gö6976, and rottlerin suppressed EGF-induced Pttg1 induction (not different from baseline), as well as EGF-induced PCNA and entry of cells into S-phase (Fig. 6Go).


Figure 6
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  		Fig. 6. Involvement of cPKCs and nPKCs in EGF-Induced Pttg1 mRNA Expression and Cell Proliferation

TtT/GF cells were serum-starved for 20 h and were subsequently treated with DMSO (0.1%), calphostin C (3 µM), Gö6976 (3 µM), or rottlerin (3 µM) for 1 h before stimulation with EGF (5 nM). Total RNA was extracted 20 h after EGF induction and Pttg1 mRNA was expression determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratio of Pttg1 mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 mRNA expression levels of all other groups were normalized to this control group. Twenty hours after EGF induction, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. Relative Pttg1 mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). *, P < 0.05. A representative experiment of three independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel).

 
Figure 7AGo demonstrates regulation of PTTG1 protein expression, as assessed by Western blotting. EGF (5 nM; 24 h) or EGF plus vehicle (0.1% dimethyl sulfoxide, DMSO) induced PTTG1 2.8 ± 0.5-fold (P < 0.001) and 2 ± 0.2-fold (P < 0.01), respectively (Fig. 7AGo). Pretreatment with AG1478 (500 nM), GF109203X (10 µM), LY294002 (10 µM), or U0126 (10 µM) prevented EGF-induced PTTG1 protein expression (P < 0.001).


Figure 7
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  		Fig. 7. Regulation of PTTG1 Protein Expression and Effect of Signaling Blockers on ERK1/2 Phosphorylation

A, Regulation of PTTG1 protein expression. TtT/GF cells were serum-starved for 20 h and were subsequently treated with DMSO (0.1%), AG1478 (500 nM), GF109203X (10 µM), U0126 (10 µM), or LY294002 (10 µM) for 1 h before stimulation with EGF (5 nM). Protein extraction was performed 24 h after EGF induction and PTTG1 protein expression was determined. The ratio of PTTG1 vs. ß-actin protein expression was calculated by densitometric analysis of each treatment group. The PTTG1/ß-actin ratio of the control group was set as 1.0. Relative PTTG1 protein expression levels of all other groups were normalized to this control group. Relative PTTG1 protein expression (mean ± SEM) of three independently performed experiments (upper panel). **, P < 0.01; ***, P < 0.001. A representative experiment of three independently performed experiments is shown (lower panel). B, Effect of signaling blockers on ERK1/2 phosphorylation. TtT/GF cells were serum-starved for 20 h and were subsequently treated with GF109203X (10 µM) or LY294002 (10 µM) for 1 h before stimulation with EGF (5 nM). Protein extraction was performed 15 min after EGF induction and phosphorylated and total ERK1/2 were determined with the use of specific antibodies. A representative experiment of two independently performed experiments is shown.

 
To identify a potential cross-talk between signaling pathways downstream of EGFR, TtT/GF cells were pretreated with the global PKC inhibitor GF109203X (10 µM) or the PI3K inhibitor LY294002 (10 µM) before stimulation with EGF (5 nM; 15 min). As shown in Fig. 7BGo, EGF-induced ERK1/2 phosphorylation was suppressed by inhibition of PI3K, whereas inhibition of PKC had no effect.

Janus Activating Kinase (JAK) Signaling Is Not Involved in EGF-Induced Pttg1 Expression and Cell Proliferation
To examine the role of the JAK/signal transducer and activator of transcription (STAT) pathway in EGF-induced Pttg1 expression and cell proliferation, TtT/GF cells were pretreated for 60 min with increasing concentrations of JAK inhibitor I (0.1–10 µM) (Fig. 8AGo). At the concentrations tested, pretreatment with JAK inhibitor I did not alter EGF-induced Pttg1 expression or cell proliferation (Fig. 8AGo). The activity of JAK inhibitor I (1 and 10 µM) was confirmed by suppression of leukemia inhibitory factor (LIF)-induced (1 nM; 60 min) suppressor of cytokine signaling-3 (SOCS-3) mRNA expression (Fig. 8BGo).


Figure 8
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  		Fig. 8. JAK Signaling Is Not Involved in EGF-Induced Pttg1 mRNA Expression and Cell Proliferation

A, TtT/GF cells were serum-starved for 20 h and were subsequently treated with increasing concentrations of JAK inhibitor I (0.1–10 µM) for 1 h before stimulation with EGF (5 nM). Eighteen hours after EGF induction, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. Cell cycle analysis of three independently performed experiments is shown (upper panel). Total RNA was extracted 20 h after EGF induction and Pttg1 mRNA expression was determined by Northern blot. A representative experiment of two independently performed experiments is shown (lower panel). B, Inhibition of LIF-induced SOCS3 mRNA expression by JAK inhibitor I. TtT/GF cells were serum-starved for 20 h and were subsequently treated with JAK inhibitor I (1 and 10 µM) for 1 h before stimulation with LIF (1 nM). Total RNA was extracted 1 h after LIF induction and SOCS3 mRNA expression was determined by Northern blot. A representative experiment of two independently performed experiments is shown.

 
Differential Regulation of Pttg1 Expression and Cell Proliferation by MAPK Signaling Pathways
Pretreatment of TtT/GF cells with increasing concentrations of the MEK inhibitor U0126 (0.1–10 µM) dose-dependently suppressed EGF-induced entry of cells into S-phase (Fig. 9AGo). EGF (5 nM; 18 h) enhanced cell entry into the S-phase approximately 3-fold (P < 0.01), and pretreatment with U0126 at a concentration as low as 1 µM suppressed this effect (P < 0.05) (Fig. 9AGo). The activity of 1–10 µM U0126 was confirmed by demonstrating suppression of both basal and pituitary adenylate cyclase-activating polypeptide (PACAP)-38-induced (50 nM; 10 min) ERK1/2 phosphorylation (Fig. 9DGo; upper panel).


Figure 9
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  		Fig. 9. MAPK Signaling: Differential Regulation of Pttg1 mRNA Expression and Cell Proliferation by ERK1/2, p38, and JNK

TtT/GF cells were serum-starved for 20 h and were subsequently treated with increasing concentrations of U0126 (0.1–10 µM) (A), SB203580 (0.2–20 µM) (B), or JNK inhibitor I (0.1–10 µM) (C) for 1 h before stimulation. A–C, Cell cycle analysis (upper panel). Eighteen hours after EGF (5 nM) induction, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. Cell cycle analysis of three independently performed experiments is shown (upper panel). *, P < 0.05; **, P < 0.01. D, Western blot—potency of MAPK inhibitors. Total protein was extracted 10 min after PACAP-38 (50 nM) (upper panel) and 15 min (middle panel) or 3 h (lower panel) after TNF{alpha} (1.8 nM) induction. Phosphorylation of ERK1/2 (upper panel) and p38 (middle panel) and c-Jun protein expression (lower panel) was determined with the use of specific antibodies. A representative experiment of two independently performed experiments is shown (lower panel). E, Pttg1 mRNA expression. Total RNA was extracted 20 h after EGF induction and Pttg1 mRNA expression was determined by Northern blot. A representative experiment of two independently performed experiments is shown.

 
In a similar dose-response curve (0.2–20 µM), the effect of SB203580, a p38 inhibitor, on EGF-induced proliferation was examined (Fig. 9BGo). Pretreatment with SB203580 inhibited EGF-enhanced cell entry into S-phase only at a concentration of 20 µM (P < 0.01) (Fig. 9BGo). The activity of lower doses of SB203580 (2 µM) was confirmed by demonstrating suppression of TNF{alpha}-induced (1.8 nM; 15 min) p38 phosphorylation (Fig. 9DGo; middle panel).

In contrast to U0126 and SB203580, pretreatment of TtT/GF cells with increasing concentrations of c-Jun N-terminal kinase (JNK) inhibitor I (0.1–10 µM) had no effect on EGF-induced proliferation (Fig. 9CGo). The activity of JNK inhibitor I was confirmed by demonstrating suppression of TNF{alpha}-induced (1.8 nM; 3 h) c-Jun expression (Fig. 9DGo; lower panel).

Similar to the proliferation studies, U0126 had a more potent suppressive effect on EGF-induced Pttg1 mRNA expression as compared with the effects of SB203580 (Fig. 9EGo), whereas pretreatment with JNK inhibitor I did not alter Pttg1 mRNA levels (Fig. 9EGo).

Kinetics of EGFR/ERK Activation and Identification of MEK-Dependent Downstream Signaling Molecules
EGF (5 nM) induced rapid and transient activation of the EGFR and phosphorylation of ERK1/2, which peaked 10 min after induction (Fig. 10AGo). ERK activation was followed by Elk-1 phosphorylation (10–30 min) and induction of c-Fos protein expression (30 min) (Fig. 10AGo). No effect was observed on Sp1 expression. Pretreatment with the MEK inhibitor U0126 (10 µM) suppressed EGF-induced ERK1/2, as well as Elk-1 phosphorylation and c-Fos expression (Fig. 10BGo).


Figure 10
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  		Fig. 10. Kinetics of EGFR/ERK Activation and MEK-Dependent Transcription Factors

TtT/GF cells were serum-starved for 20 h and were subsequently treated with EGF (5 nM) for the indicated times (A). Pretreatment with U0126 (10 µM) was performed for 45 min, followed by coincubation with EGF (5 nM) for 15 min (B). Expression of phosphorylated EGFR (p-EGFR), phosphorylated ERK1/2 (p-ERK1/2), and total ERK1/2 (loading control) was performed in whole-cell extracts (upper three lanes), whereas expression of phosphorylated Elk-1 (p-Elk-1), c-Fos, SP1, and ß-actin (loading control) was performed in nuclear-enriched extracts (lower four lanes). A representative experiment of two independently performed experiments is shown.

 
Pttg1 and PCNA mRNA Expression in Relation to the Cell Cycle
To characterize the temporal relationship of EGF-induced Pttg1 and PCNA mRNA in relationship to the cell cycle, RNA extraction and cell cycle analysis were performed at 2-h intervals for up to 26 h after EGF induction (Fig. 11AGo). PCNA mRNA abundance peaked early at approximately 14 h, followed by the peaking of S-phase between 14 and 18 h. However, Pttg1 mRNA levels peaked later (~18 h after stimulation), at the S-G2 transition, to decline thereafter (Fig. 11AGo). Figure 11BGo shows decreased PTTG1 protein levels after completion of the cell cycle (26–30 h). Thus, EGF-induced PCNA peaks early in S-phase, whereas Pttg1 achieved maximal levels later, at the S-G2 transition (Fig. 11AGo). These results indicate that Pttg1 mRNA induction by EGF is closely related to cell cycle progression. To further confirm this, cells were blocked in early S-phase by pretreatment with aphidicolin for 60 min (Fig. 12Go). EGF enhanced Pttg1 mRNA levels (2.5 ± 0.6-fold) (P < 0.001), whereas aphidicolin pretreatment dose-dependently suppressed this effect (Fig. 12Go).


Figure 11
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  		Fig. 11. EGF-Induced Pttg1 and PCNA mRNA Expression in Relationship to the Cell Cycle

TtT/GF cells were serum-starved for 20 h and were subsequently treated with EGF (5 nM) for indicated times. A, Time-dependent effects on Pttg1 mRNA expression. Total RNA was extracted at the times indicated and Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PBF and PCNA, respectively. The ratios of Pttg1 and PCNA mRNA vs. ß-actin mRNA were calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio or PCNA/ß-actin ratio of the control group was set as 1.0. Relative mRNA expression levels of other groups were normalized to these control groups. Relative Pttg1 and PCNA mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). *, P < 0.05; **, P < 0.01. B, Reduction of PTTG1 protein expression after completion of the cell cycle. Total protein was extracted at the times indicated and PTTG1 protein expression was performed by Western blot. A representative experiment of two independently performed experiments is shown.

 

Figure 12
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  		Fig. 12. Effect of Early S-Phase Blockade on Pttg1 mRNA Expression

TtT/GF cells were serum-starved for 20 h and were subsequently treated with aphidicolin for 1 h before stimulation with EGF (5 nM). Total RNA was extracted 20 h after EGF induction. Pttg1 mRNA expression was determined by Northern blot. Subsequently, membranes were stripped and reblotted with specific probes for PCNA and then for ß-actin. The ratio of Pttg1 mRNA vs. ß-actin mRNA was calculated by densitometric analysis of each treatment group. The Pttg1/ß-actin ratio of the control group was set as 1.0. Relative Pttg1 mRNA expression levels of all other groups were normalized to this control group. At the indicated times, cells were fixed and cell cycle analysis was performed by flow cytometry. Percentage of cells in G0/1-phase is depicted by black bars, cells in S-phase by white bars, and cells in the G2/M-phase of the cell cycle by gray bars. Relative Pttg1 mRNA expression (mean ± SEM) of three independently performed experiments (upper panel). **, P < 0.01; ***, P < 0.001. A representative experiment of three independently performed experiments is shown (middle panel). Cell cycle analysis of three independently performed experiments (lower panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study demonstrates that pituitary TtT/GF FS cells express PTTG1 and that EGFR ligands such as EGF and TGF{alpha} induce PTTG1 expression in a cell cycle-dependent manner. Signaling molecules involved in the EGFR-mediated mitogenic response and associated PTTG1 expression include PI3K, PKC, and the MAPK ERK1/2.

PTTG1 is overexpressed in most pituitary tumors (11); however, little is known of PTTG1 expression and regulation within different anterior pituitary cell populations. RT-PCR revealed expression of both Pttg1 and its binding factor PBF in TtT/GF pituitary FS cells, as well as the EGFR.

EGF is mitogenic for corticotropes (7, 33) and gonadotropes (34, 35) and induces transdifferentiation of somatotropes to lactotropes in some tumor cell models (36, 37). EGFR is abundantly expressed in the normal pituitary (38) and, despite initial conflicting reports (39, 40), EGFR appears to be expressed in most pituitary tumors at varying levels (8, 9, 41, 42, 43, 44). EGF induces PTTG1 in astrocytes (45) and is herein shown to induce Pttg1 mRNA in pituitary TtT/GF cells. EGF-induced Pttg1 expression was associated with increased cell proliferation, evidenced by elevated PCNA levels and enhanced entry of cells into S-phase. Expression of the PTTG1 binding factor PBF was not altered by EGF, indicating alternative mechanisms for PBF mRNA regulation. The EGFR ligand TGF{alpha} demonstrated a similar stimulation pattern for Pttg1 expression and cell proliferation, and the specificity of EGFR signaling was confirmed by use of EGFR antagonists.

Binding of EGF to EGFR triggers receptor dimerization, leading to activation of multiple intracellular signaling pathways (46). To explore the underlying signaling mechanisms in FS cells, we employed pharmacological blockade of transduction pathways specifically implicated in EGFR signaling in other cellular systems (47, 48, 49, 50). Our results demonstrate a particular involvement of MAPK and PI3K, as well as at least partial involvement of both cPKC and nPKC isoforms for EGF-induced Pttg1 mRNA expression and cell proliferation, as well as PTTG1 protein expression. In contrast, the classical JAK/STAT signaling appears not to be involved in this EGFR-mediated biological response. However, possible JAK-independent involvement of STAT proteins through EGFR-mediated Src kinase activation and subsequent STAT phosphorylation (51) or direct EGFR-STAT interaction cannot be excluded.

Because EGF activation and associated mitogenic responses can be mediated by distinct MAPK subfamily members depending on the particular cell model (52, 53), we employed specific inhibitors of MEK1/2, as well as p38 and JNK, to further characterize the involvement and relative potency of MAPK subgroups. The activity of the agents used (U0126, SB203580, and JNK Inhibitor I) was confirmed by demonstrating inhibition of PACAP-38-induced ERK1/2 phosphorylation and TNF{alpha}-induced p38 phosphorylation and c-Jun expression, respectively. U0126 suppressed EGF-induced cell proliferation at the lowest active concentration, and SB203580 inhibitory effects were only achieved at higher concentrations, whereas JNK inhibition did not alter EGF-induced cell proliferation or EGF-induced Pttg1 mRNA expression. Thus, in FS TtT/GF cells, ERK1/2 appears to be the primary MAPK signaling pathway involved in the EGFR-mediated response. Furthermore, partial suppression of EGF-induced ERK phosphorylation by the PI3K (but not PKC) inhibitor suggests a crosstalk between PI3K and ERK signaling downstream of EGFR, as has been reported in mesangial cells (54). Kinetic analysis of EGFR/ERK1/2 activation by EGF demonstrated a rapid but transient activation of the receptor and ERK1/2, followed by MEK-dependent activation of transcription factors Elk-1 and c-Fos. Thus, Elk-1 and c-Fos appear to be involved in the early and MAPK-dependent response to mitogenic induction with EGF. However, SP1, an ubiquitous transcription factor, which often shows an ERK-dependent expression pattern (55), was not induced by EGF.

A critical function of PTTG1 is that of a mammalian securin protein, regulating sister chromatid separation during mitosis (25). PTTG1/securin is known to be mainly regulated at the posttranscriptional level, and its rapid degradation by the anaphase-promoting complex at the end of metaphase releases tonic separase inhibition (25). Because of the highly oncogenic potential of PTTG1, it is essential to understand the mechanisms that control PTTG1 transcription in normal and transformed tissues. Treatments with estrogen (56) or insulin (57) induce PTTG1 mRNA in particular models, but in most cases it has been unclear whether PTTG1 transcription was a direct effect or secondary to induction of cell proliferation and associated activation of the cell cycle machinery. We therefore characterized the temporal relationship of EGF-induced Pttg1 and PCNA mRNA expression during cell cycle progression and showed that PCNA peaked early in S-phase, whereas Pttg1 mRNA achieved maximal levels about 18 h after induction at the S-G2 transition. Although Pttg1 mRNA levels declined thereafter, they still remained elevated compared with controls, probably due to the long Pttg1 mRNA half-life (>8 h as determined by experiments with actinomycin D treatment; data not shown). Decreased PTTG1 protein expression after completion of one cell cycle was confirmed by Western blot, consistent with the reported degradation at the end of the metaphase (25). To confirm the cell cycle-dependency of Pttg1 transcription, cells blocked in early S-phase by aphidicolin showed abrogated EGF-induced Pttg1 expression, confirming the relationship of EGF-induced Pttg1 mRNA expression to cell cycle progression. Thus, this study in pituitary FS cells suggests that regulation of Pttg1 mRNA expression is mainly cell cycle-dependent, because it exhibits a specific expression pattern during cell cycle progression in synchronized cells and is suppressed by pharmacological blockade of cells in a particular cell cycle phase. High levels of PTTG1 mRNA in transformed cells may occur as a result of increased cell proliferation; however, additional mechanisms may be involved particularly in transformed tissue, which may release baseline control of PTTG1 transcription, because pituitary tumors that exhibit high PTTG1 mRNA expression are not necessarily highly proliferative. Such mechanisms remain to be elucidated.

In summary, pituitary TtT/GF FS cells express EGFR, enabling these cells to respond to EGFR ligands such as EGF and TGF{alpha}, thereby inducing cell cycle-dependent Pttg1 expression. Growth factors are abundant within the pituitary microenvironment and may sustain the growth of pituitary adenomas by autocrine/paracrine feedback. EGF, which is known to act directly on hormone-secreting pituitary cells (7, 33, 34, 35, 36, 37), is herein shown to effect cell proliferation and Pttg1 expression in hormonally inactive FS cells, the major source of pituitary cytokines and growth factors (3, 4, 5, 6). Because enhanced PTTG1 expression induces paracrine secretion of growth factors (21, 23, 58, 59), this mechanism may contribute to the paracrine tumorigenic pituitary microenvironment.

Characterization of intracellular signaling mechanisms involved in growth factor-induced PTTG1 expression will allow specific targeting of PTTG1 abundance in pituitary FS cells. Pituitary adenomas could benefit from inhibition of EGFR-mediated PTTG1 expression in FS cells by intracellular blockade of paracrine signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
DMEM, fetal bovine serum, penicillin/streptomycin, and amphotericin B were purchased from Invitrogen (Carlsbad, CA). Plasticware for cell culture was purchased from Corning Inc. (Corning, NY). EGF and TGF{alpha} were obtained from Sigma (St. Louis, MO). LIF was obtained from Chemicon International (Temecula, CA). GF109203X was purchased from Biomol (Hamburg, Germany). PACAP-38 was from Bachem (Torrance, CA), and murine TNF{alpha} was from R&D Systems (Minneapolis, MN). AG1478, LY294002, aphidicolin, rottlerin, Gö6976, calphostin C, JAK inhibitor I, SB203580, and JNK inhibitor I were obtained from Calbiochem (San Diego, CA). U0126 was from Promega (Madison, WI), and gefitinib was purchased from Biaffin GmbH & Co. (Kassel, Germany). Doses for inhibitors were derived from the literature (5, 60, 61, 62, 63, 64, 65, 66, 67, 68).

Cell Culture
TtT/GF cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin/streptomycin, and 0.4% amphotericin B in a 5% CO2 atmosphere. Cells were plated in 100-mm dishes at a density of approximately 1.6 x 106 cells in complete medium for 24–48 h and were synchronized by serum starvation (medium with 0.2% BSA) for an additional 20 h. Treatment agents were added with fresh serum-depleted medium, and samples were collected at the indicated times.

RT-PCR and Sequencing
RNA extraction was performed using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription of 1.0 µg total RNA derived from FS TtT/GF cells was performed with Moloney murine leukemia virus-Reverse Transcriptase (Invitrogen). PCR of 2 µl cDNA sample was performed in a total reaction volume of 50 µl containing 10x PCR buffer [1x: MgCl2 (1.5 mM), DMSO (5%), 0.2 mM of each deoxynucleoside triphosphate, 2.5 U Taq DNA polymerase (Invitrogen) and 0.5 µM sense and antisense primers (Invitrogen)]. After initial denaturation (94 C, 2 min), 35–40 PCR cycles followed (denaturation 94 C, 30 sec; annealing at appropriate temperature, 30 sec; extension 72 C, 1 min) and the reaction was terminated by a single elongation step at 72 C for 10 min. A 566-nt PCR product of murine Pttg1 (nt 312–878; GenBank accession no. NM_013917) was generated with a specific primer pair (sense, 5'-ATGGCTACTCTTATCTTTGTTGAT-3'; and antisense, 5'-TCATGTGACAAGTTGCTTACAGC-3') at an annealing temperature of 56 C. A 292-nt PCR product of murine PBF (nt 121–413; GenBank accession no. AK089887) was created with a specific primer pair (sense, 5'-CGCGCAGGAACCTCCGAGAGTGG-3'; and antisense, 5'-GCTTCCGGCTCTTCTTCCAGCGG-3') at an annealing temperature of 56 C. A 319-nt PCR product of murine full-length EGFR was generated as described previously (69). Electrophoresis of PCR products was performed on a 1.0% agarose gel and specific bands gel-purified with QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA), and cycle sequencing performed by the University of California, Los Angeles Sequencing Core (Los Angeles, CA).

Templates for Probes and Northern Blot Analysis
Probe template for murine PBF was generated by PCR (described above). Probes for murine Pttg1, PCNA, and SOCS3 were generated as described (70, 71, 72). The ß-actin probe was a 1.076-kb fragment of the mouse ß-actin gene (Ambion, Austin, TX). RNA extraction was performed using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Northern blot analysis was performed as described (5). In brief, 10–20 µg of total RNA were electrophoresed on a 1% agarose, 6.4% formaldehyde gel; transferred to a Hybond-n + membrane (Amersham, Arlington, IL); and UV-crosslinked. Probes were labeled with [{alpha}-32P]CTP using the Prime-It Random Primer Labeling Kit (Stratagene, La Jolla, CA). Micro Bio-Spin Chromatography Columns (Bio-Rad, Hercules, CA) were used to purify probes. Membrane prehybridization and hybridization were performed using QuickHyb Solution (Stratagene) and then exposed to Hyperfilm MP (Amersham) for 1–4 d at –70 C.

Protein Extraction and Western Blotting
For Western blot experiments, approximately 1.6 x 106 cells were seeded in 100-mm dishes and grown for 24–48 h in 5 ml complete medium, followed by incubation with serum-free medium (DMEM with 0.2% BSA) for 20 h. Treatments were added in fresh serum-depleted medium, and samples were collected at the indicated times. For whole-cell protein extraction, cells were lysed in 500 µl ice-cold lysis buffer (pH 7.6) containing 0.5 mM dithiothreitol, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100, and protease inhibitors [1 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 100 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride]. Lysates were centrifuged at 13,000 x g for 20 min at 4 C, and protein concentration in supernatants was determined by Bradford’s method (Bio-Rad, Richmond, CA). Nuclear extractions were performed with NE-PER Nuclear and Cytoplasmatic Extraction Reagents according to the instructions of the supplier (Pierce Biotechnology, Rockford, IL). Western blot analysis was performed according to the guidelines of NuPAGE electrophoresis system protocol (Invitrogen). In brief, samples (50 µg protein per lane) were heated for 10 min at 70 C. Proteins were separated on NuPAGE 4–12% Bis-Tris gels and electrotransferred for 1 h onto polyvinylidene difluoride (Invitrogen). Membranes were blocked for 1 h in 2% nonfat dry milk in TBS-T (0.1% Tween 20 in Tris-buffered saline) buffer and incubated overnight with primary antibody. The following primary antibodies were used: rabbit anti-PTTG1 antibody (1:100; Zymed, San Francisco, CA), mouse anti-pERK1/2, rabbit anti-ERK1/2 (1:200), goat anti-c-Jun antibody (1:10,000), mouse anti-c-Fos, rabbit anti-Sp1, mouse anti-p-p38 (1:5000; Sigma), mouse anti-p-Elk-1 (1:2000; Cell Signaling), and mouse anti-ß-actin (1:1000; Sigma). After washing with TBS-T, membranes were incubated with peroxidase-conjugated secondary antibody for 2 h (2% nonfat dry milk in TBS-T buffer). Blots were washed, and hybridization signals were measured by ECL detection system (Amersham).

Flow Cytometric Cell Cycle Analysis
After synchronization by incubation with serum-free medium (DMEM with 0.2% BSA) for 20 h, treatments were added in fresh serum-depleted medium and samples collected at the indicated times. Cells were washed and fixed in 50% ice cold ethanol. After centrifugation, cell pellets were treated with 5 µg/ml propidium iodide solution (Sigma) containing 100 µg/ml ribonuclease (Becton Dickinson, Mountain View, CA) for 1 h, and samples were analyzed in a FACScalibur system (Becton Dickinson) using CellQuest software.

Statistical Analysis
The percentage of cell cycle phases was analyzed by ModFit LT software (version 2.0; Becton Dickinson). NIH Image 1.59 software was used for densitometric analysis of specific bands in Northern and Western blots. Statistical analysis was performed with two-way ANOVA (Figs. 2AGo, 3AGo, and 11AGo) or one-way ANOVA (remaining figures) using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). Results are expressed as mean ± SEM of independently performed experiments, and P < 0.05 was considered significant.


   FOOTNOTES
 
This study was supported by a scholarship from the Deutsche Forschungsgemeinschaft (VL 55/1-1), by National Institutes of Health Grant CA 075979 (to S.M.), and by the Endocrine Fellows Foundation.

Disclosure statement: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: cPKC, Classical PKC; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; FS, folliculostellate; JAK, Janus activating kinase; JNK, c-Jun N-terminal kinase; LIF, leukemia inhibitory factor; MEK, MAPK kinase/ERK kinase; nPKC, novel PKC; nt, nucleotide; PACAP-38, pituitary adenylate cyclase-activating polypeptide; PBF, PTTG binding factor; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3 kinase; PKC, protein kinase C; PTTG1, pituitary tumor transforming gene-1; SOCS-3, suppressor of cytokine signaling-3; STAT, signal transducer and activator of transcription.

Received for publication July 7, 2006. Accepted for publication August 28, 2006.


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