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Molecular Endocrinology 18 (6): 1558-1569
Copyright © 2004 by The Endocrine Society

Activin Inhibits Pituitary Prolactin Expression and Cell Growth through Smads, Pit-1 and Menin

Annie Lacerte, Eun-Hye Lee, Rachel Reynaud, Lucie Canaff, Chantal de Guise, Dominic Devost, Suhad Ali, Geoffrey N. Hendy and Jean-Jacques Lebrun

Hormones and Cancer Research Unit, Department of Medicine (A.L., E.-H.L., R.R., C.G., D.D., S.A., J.-J.L.), and Departments of Physiology and Human Genetics (L.C., G.N.H.), Royal Victoria Hospital, McGill University, Montreal, Quebec, H3A 1A1, Canada

Address all correspondence and requests for reprints to: Dr. J. J. Lebrun, Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, H7.81, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1, Canada. E-mail: JJ.Lebrun{at}MUHC.McGill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activin, a member of the TGFß superfamily, is a negative regulator of cell growth and prolactin (PRL) production in pituitary lactotrope cells. However, the mechanisms by which this growth factor exerts its growth-inhibitory and -repressive effect on PRL remain unclear. In this study, we show that activin negatively regulates PRL expression at the transcriptional level through the Smad pathway and the multiple endocrine neoplasia type 1 gene product, menin. Our results also demonstrate that the tumor suppressor menin is required for activin-induced growth arrest of somatolactotrope cells. Moreover, we show that activin represses transcription and expression of Pit-1, a pituitary transcription factor that is essential for maintenance and development of lactotrope cells. We defined two Pit-1 DNA-binding sites in the proximal region of the PRL promoter as critical for the activin-mediated inhibition. Together, our results highlight the Smad pathway and the tumor suppressor menin as key regulators of activin effects on PRL and Pit-1 expression, as well as on cell growth inhibition, and emphasize the critical role of activin in the regulation of pituitary function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROLACTINOMAS ARE THE major type of human secretory pituitary tumors. In addition to their hyperproliferative capacity, these cells secrete large amounts of prolactin (PRL) causing severe endocrine and reproductive disorders. Despite the high incidence of this type of tumors, the molecular basis for development of these pituitary disorders remains unknown.

Pituitary gland function is controlled by a large array of hormones and growth factors. Activin, a member of the TGFß family, regulates the secretion of a variety of endocrine products (1) and plays an important role in regulating anterior pituitary gland function. Activin was first isolated from the gonads because of its ability to stimulate pituitary FSH synthesis and secretion from the gonadotropes (2). The pituitary action of activin is not restricted to gonadotropes, and activin also modulates the function of other pituitary cell types such as the somatotropes and lactotropes. In addition to stimulating FSH release from the gonadotropes, activin inhibits basal GH and ACTH secretion (2). Finally, activin acts as a negative regulator of PRL expression and secretion in pituitary primary culture and cell lines (3). In addition, activin regulates cell growth and differentiation of numerous cell types. The antiproliferative and proapoptotic effects of activin have been observed in many different cell types such as erythroleukemia (4), capillary endothelial (5), immune (6, 7), breast cancer (8, 9, 10), and hepatocytes (11, 12, 13, 14, 15). Consistent with the critical role of activin in cell growth regulation, alterations of the activin signaling pathway, such as mutation or truncation of the activin receptor, are associated with human tumors (16, 17).

Activin signals through a complex of two transmembrane serine/threonine kinase receptors (type I and type II receptors) (4, 18). Upon ligand binding, the type II receptor phosphorylates the type I receptor, and the activated receptor complex then recruits and phosphorylates the receptor-regulated Smad2 and Smad3 (18, 19, 20, 21). These C-terminally phosphorylated Smads then undergo a change in conformation, which results in dissociation from the receptor complex and association with the common-partner Smad4 (22, 23, 24). The Smad complex translocates into the nucleus where it regulates transcription of target genes through DNA binding and functional recruitment of specific transcription factors and coregulators, thus providing tissue specificity (25, 26). However, the mechanisms and signaling pathways by which activin regulates lactotrope pituitary cell function and growth are not well characterized.

An important player in regulating both PRL and GH expression is the pituitary-specific transcription factor Pit-1 (GH factor-1) (27, 28). Pit-1 is required for the generation and maintenance of three cell types (lactotropes, somatotropes, and thyrotropes) in the anterior pituitary gland (29). Although activin was reported to apparently not modulate Pit-1 mRNA levels (30), it was shown to induce Pit-1 degradation in MtTW15 somatotrope cells (31). However, the mechanisms by which activin controls Pit-1 activity are still not fully determined.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary, especially prolactinomas (32). Interestingly, overexpression of menin, the product of the MEN1 gene, leads to reduced PRL expression (33). Moreover, inactivation of menin expression suppresses TGFß-induced growth inhibition (34). We recently showed that menin physically interacts with Smad3 in somatolactotrope cells, and its inactivation blocks TGFß signaling (34). However, such a role for menin downstream of the activin receptor signaling pathway remains to be determined.

The multiple effects of activin on hormone production and secretion highlight the important role played by this growth factor in regulating pituitary function. However, the mechanisms remain unclear. In this study, we show that activin negatively regulates PRL gene transcription and that the Smad pathway is involved in the mediation of this effect. Moreover, we show, for the first time, that menin is required for activin-mediated inhibition of PRL expression. We also show that activin-mediated inhibition of PRL expression occurs through reduction of Pit-1 expression, and we define two Pit-1 sites in the proximal region of the PRL gene promoter as critical to activin-mediated PRL inhibition. Finally, we found that menin is also important for activin-induced cell growth inhibition in somatolactotrope cells. Together, our results clearly demonstrate a critical role for the growth factor activin in regulating inhibition of pituitary cell growth and Pit-1/PRL expression through the Smads and menin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activin Inhibits PRL Gene Transcription
To first characterize the role of activin on PRL gene expression, we used rat somatolactotrope GH4C1 cells, a highly differentiated neuroendocrine cell line that retains the capacity to synthesize and secrete GH and PRL in a hormone-regulated manner (35, 36). The GH4 cells were established from rat pituitary tumor cells and are widely used as an in vitro model of pituitary tumors (37, 38). As shown in Fig. 1AGo (upper panel), PRL mRNA levels were markedly reduced after 4 h of activin treatment and had returned to basal levels by 24 h. Reprobing of the stripped membrane for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA showed equal loading (Fig. 1AGo, lower panel). To then evaluate whether the effect of activin on PRL mRNA was followed by a decrease in PRL protein expression, extracts of GH4C1 cells treated or not with activin for different periods of time were analyzed by Western blot. As shown in Fig. 1BGo (upper panel), activin clearly inhibited PRL expression by 16 h to reach a maximum inhibition by 24 h. Reprobing of the membrane for signal transducer and activator of transcription (Stat)3 protein showed equal loading (Fig. 1BGo, lower panel). The data indicate that activin blocks PRL gene expression. The same results were obtained using another pituitary-derived PRL-expressing tumor cell line (GH3), thus strengthening the conclusion (data not shown).



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  		Fig. 1. Transcriptional Regulation of PRL Gene Promoter by Activin

A, Total RNA from GH4C1 cells stimulated with activin for the indicated periods of time were analyzed by Northern blot using specific probes for PRL and GAPDH. B, Whole-cell lysates from GH4C1 cells stimulated with activin for the indicated periods of time were analyzed by Western blot using anti-PRL antibody. The membrane was reprobed with an anti-Stat3 antibody for loading control. C and D, GH4C1 cells were transfected with the rPRL-3kb or the hPRL-5kb (panel C) or the indicated truncated promoter constructs (panel D) with a ß-galactosidase expression plasmid, and the activin response was measured by luciferase assay 18 h after stimulation. *, P < 0.05 compared with no activin treatment (all panels).

 
To determine whether activin directly regulates PRL expression at the transcriptional level, GH4C1 cells were transfected with reporter constructs containing either 3 kb of the rat PRL promoter fused with the luciferase gene (rPRL-3kb) (39) or 5 kb of the human PRL promoter (hPRL-5kb) (40) and stimulated or not with activin for 16 h. As shown in Fig. 1CGo, activin stimulation led to a 50% reduction in luciferase activity compared with control. Thus, activin exerts a direct transcriptional effect on PRL gene expression.

The PRL gene promoter consists of distal and proximal regions that both contain important regulatory sequences. To explore further the promoter sequences mediating activin down-regulation of PRL expression, constructs containing both distal and proximal regions of the rat (rPRL-3kb) and human (hPRL-5kb) PRL gene promoters and constructs containing only the proximal elements (rPRL-450bp and hPRL-250bp) fused to the luciferase reporter gene were used. The constructs were transfected into GH4C1 cells, and the luciferase activity was measured after activin treatment. As shown in Fig. 1DGo, removal of the distal element of both rat and human promoters did not affect PRL-induced repression (rPRL-450bp, hPRL-250bp). The results indicate that activin mediates its inhibitory effect on PRL gene transcription through the proximal region of the PRL gene promoter.

Smad Pathway Is Critical for Inhibition of PRL Gene Transcription by Activin
The Smad pathway represents the canonical pathway downstream of activin receptors. Activin signaling via activation of Smads was demonstrated by immunoblot analysis using a specific antibody to phosphoSmad2. Activin treatment of GH4C1 cells led to a rapid increase in endogenous Smad2 phosphorylation (Fig. 2AGo, upper panel). The membrane was stripped and reprobed with an anti-Stat3 antibody to show equal loading (Fig. 2AGo, lower panel).



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  		Fig. 2. The Smad Pathway Is Critical for Activin Inhibition of PRL Gene Promoter Activity

A, GH4C1 cells were treated with activin (0–60 min.) Whole-cell lysates were analyzed by Western blot using a specific antibody to phospho-Smad2 (upper panel). The membrane was stripped and reprobed with an anti-Stat3 antibody as a loading control. B, GH4C1 cells were transfected with the PRL-3kb reporter construct, the ß-galactosidase expression plasmid, and the different Smad expression plasmids, as indicated. Cells were then stimulated with activin and assessed for luciferase activity. *, P < 0.05 compared with no activin treatment.

 
To investigate the role of the Smads in the activin-induced PRL inhibition, we used the inhibitory Smad7 (41), the dominant-negative forms of Smad2 (DNSmad2) (23), and Smad3 (DNSmad3) (42) in which the serine residues within the motif SSXS, the target of Smad phosphorylation by the type I receptor, are mutated to alanine. GH4C1 cells were transiently cotransfected with the 3-kb PRL gene promoter reporter construct and the cDNA encoding for either Smad7, DNSmad2, or DNSmad3. As shown in Fig. 2BGo, the activin-induced decrease in luciferase activity observed in control cells is blocked in GH4C1 cells overexpressing Smad7 or the dominant negative Smad2 or 3. These results clearly demonstrate the requirement of the Smad pathway in activin-mediated inhibition of PRL gene promoter activation.

Menin Is Required for Activin-Mediated Inhibition of PRL Expression
Interestingly, a recent study showed that overexpression of menin in GH3 pituitary cells inhibits PRL gene expression (33). To explore the function of menin in activin signaling, GH4C1 cells were treated with either antisense oligonucleotides to the 5'-coding sequence of menin (AS-oligo) or a scrambled sequence as a control (C-oligo). To validate our system, we examined the ability of the antisense oligonucleotide to suppress menin expression. As shown in Fig. 3AGo, treatment of GH4C1 cells with the AS-oligo significantly inhibited menin protein expression by 75% as compared with the C-oligo. Densitometric analysis was performed using Fluorochem 8000 software that allows normalization and quantitative analysis of chemiluminescence under nonsaturating condition. To then examine the effects of menin inactivation on activin-mediated PRL inhibition, GH4C1 cells were treated with AS-oligo or C-oligo before being stimulated or not with activin. Total cell lysates were then analyzed by Western blotting using antibodies to PRL or Stat3. As shown in Fig. 3BGo, the inhibitory effect of activin on PRL protein expression is abolished in the presence of AS-oligo, but not with the C-oligo.



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  		Fig. 3. Activin-Mediated PRL Inhibition Requires Menin

A and B, GH4C1 cells were cultured in the presence of menin antisense or control oligonucleotides and stimulated or not with activin for 24 h. Cell lysates were analyzed by Western blot using antibodies to menin, PRL, and Stat3 (as a loading control). C, Total RNA from GH4C1 cells transfected with menin (M10) or GAPDH siRNA were analyzed by RT-PCR. Total cell lysates from GH4C1 cells transfected with menin (M10) or GAPDH siRNA were analyzed by Western blot using antimenin and anti-Stat3 antibodies. D, GH4C1 cells transfected with M10 or GAPDH siRNA were cultured with or without activin for 24 h. Cell lysates were analyzed by Western blot using anti-PRL and antimenin antibodies. E, GH4C1 cells were transfected with the PRL-3kb reporter construct and the ß-galactosidase expression plasmid. Cells were treated with menin antisense or control oligonucleotides and stimulated or not with activin for 18 h before being assessed for luciferase activity. *, P < 0.05 compared with no activin treatment. F, GH4C1 cells were cultured with activin for the indicated period of time. Protein extracts were analyzed by Western blot using a specific antibody to menin. The membrane was stripped and reprobed with an anti-Stat3 antibody.

 
To further support the essential role of menin in the regulation of PRL, we generated and tested three different siRNAs corresponding to the rat MEN1 gene. We used, as a control, an unrelated siRNA specific for GAPDH gene. As illustrated in Fig. 3CGo, transfection of MEN1 or GAPDH siRNA in GH4C1 cells efficiently reduced their respective mRNA and protein expression levels (55% inhibition). We chose to use M10 siRNA for subsequent experiments because this particular small interfering RNA (siRNA), which correspond to nucleotides 10–21 of the rat MEN1 coding sequence, was the most efficient in blocking menin mRNA expression. As shown in Fig. 3DGo, activin-mediated PRL expression inhibition was blocked in GH4C1 cells transfected with M10 siRNA but not affected in control cells or cells transfected with the GAPDH siRNA. Thus, reducing menin expression levels using siRNA or antisense approach, is sufficient to block activin-mediated PRL inhibition, demonstrating the critical role played by menin in activin signal transduction in pituitary cells.

We then analyzed the role of menin in activin-induced repression of the PRL gene promoter. For this, GH4C1 cells transfected with the 3-kb rat PRL gene promoter construct were treated with the menin antisense or control oligonucleotide, and cells were stimulated or not with activin. Interestingly, activin effects on the PRL gene promoter were completely blocked in the absence of menin but not affected by the control oligonucleotide (Fig. 3EGo). Therefore, menin is critical and required to mediate activin effects on inhibition of PRL expression in anterior pituitary cells. We also examined the role of activin on menin expression. As shown in Fig. 3FGo, activin clearly induces an increase in menin expression in GH4C1 cells. As menin is required to mediate the activin effects (Fig. 3Go, B–E), this suggests that it may act in a positive feedback loop to transduce activin signaling.

Activin-Dependent Down-Regulation of PRL Expression Is Pituitary Cell Specific
Production of PRL is not limited to the pituitary, and other sites such as the mammary gland (43) have been shown to produce PRL. Interestingly, the human breast cancer cell line T47D, which produces PRL (44), is also responsive to activin (10). However, as shown in Fig. 4AGo, no change in PRL expression (upper panel) was observed in response to activin in these cells. The activin responsiveness of the cells was demonstrated by the clear increase in phospho-Smad2 in response to activin (Fig. 4BGo). Moreover, Chinese hamster ovary (CHO) cells, transiently transfected with the rPRL-3kb or rPRL-450bp gene promoter constructs, showed no decrease in luciferase activity in response to activin (Fig. 4CGo). As a positive control, cells were transfected with the activin responsive promoter construct (3TPLux). Together, these data indicate that a pituitary cell-specific factor is involved in the activin-mediated down-regulation of PRL mRNA expression.



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  		Fig. 4. A Pituitary Cell-Specific Factor Is Involved in the Activin-Dependent Down-Regulation of PRL mRNA

A, Breast cancer T47D cells were treated or not with activin for various times, as indicated. Cell lysates were analyzed by immunoblot using antibodies specific to PRL or Stat3 as control. B, T47D cells were stimulated with activin for 0, 15, 30, and 60 min. Protein extracts from these cells were then subjected to Western blot analysis with an anti-phosphoSmad2-specific antibody. The membrane was stripped and reprobed with an anti-Smad2/3-specific antibody. C, CHO cells were transfected with the PRL-3kb, the PRL-450bp, or the 3TP-lux reporter construct as a positive control with the ß-galactosidase expression vector. Cells were then stimulated or not with activin and assessed for luciferase activity. *, P < 0.05 compared with no activin treatment.

 
Pit-1 Gene Transcription and mRNA and Protein Levels Are Down-Regulated by Activin
Analysis of both the rat and human PRL promoter-proximal region reveals three Pit-1 response elements, suggesting that activin-mediated PRL inhibition may be mediated via regulation of the pituitary-specific transcription factor Pit-1. This prompted us to analyze the regulation of Pit-1 gene expression by activin in GH4C1 cells. We first examined the activin effects on Pit-1 mRNA levels in GH4C1 cells. As shown in Fig. 5AGo, Northern blotting analysis of activin-treated GH4C1 cells indicated that Pit-1 mRNA levels were significantly reduced starting at 4 h to reach a maximum at 16 h after activin treatment. Pit-1 levels then returned to normal by 24 h. To then determine whether this decrease in Pit-1 mRNA was followed by a decrease in Pit-1 protein expression, GH4C1 cells stimulated with activin for different periods of time were analyzed by Western blot. As shown in Fig. 5BGo, both PRL and Pit-1 protein levels were strongly reduced upon activin treatment. To evaluate the role of menin in activin-mediated Pit-1 down-regulation, GH4C1 cells transfected with the menin siRNA (M10) or GAPDH siRNA were stimulated or not with activin, and the level of Pit-1 protein expression was evaluated using a specific anti-Pit-1 antibody. As shown in Fig. 5CGo, in the presence of menin siRNA, but not with GAPDH siRNA, activin lost its ability to repress Pit-1 protein expression, similar to that observed for PRL (Fig. 3CGo), thus indicating that menin is required for activin to block Pit-1 protein expression.



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  		Fig. 5. Activin Down-Regulates Pit-1 Gene Transcription and mRNA and Protein Levels

GH4C1 cells were cultured with activin for the indicated periods of time. A, Total RNA from GH4C1 cells stimulated with activin for the indicated periods of time were analyzed by Northern blot using specific probes for Pit-1 and GAPDH. B, GH4C1 cells were cultured with activin for the indicated periods of time. Cell lysates were immunoblotted using anti-PRL and anti-Pit-1 antibodies. Stripping and reprobing the blot with an anti-Stat3 antibody confirmed equal loading. C, Cell lysates from GH4C1 cells transfected with menin (M10) or GAPDH siRNAs and treated or not with activin were immunoblotted using anti-Pit-1 and anti-stat3 antibodies. D, GH4C1 cells were transfected with the Pit-1-lux reporter construct together with the ß-galactosidase expression plasmid with or without Smad7 expression construct. Cells were treated with activin for 18 h, and luciferase assays were performed. *, P < 0.05 compared with no activin treatment.

 
Furthermore, in GH4C1 cells transfected with a construct in which the Pit-1 gene promoter drives a luciferase reporter gene (45), activin caused a 50% inhibition of luciferase activity, and this effect was blocked in cells expressing Smad7 (Fig. 5DGo). These data indicate that activin modulates Pit-1 mRNA and protein expression in a Smad- and menin-dependent manner through inhibition of the Pit-1 gene promoter.

Pit-1 Response Elements in the PRL Gene Proximal Promoter Are Critical for Activin-Mediated Repression
The proximal PRL gene promoter region contains three Pit-1 sites (Fig. 6AGo). These sites are involved in both positive and negative regulation of PRL gene promoter activity (46). To investigate the relative importance of these sites in mediating activin repression of the PRL promoter, four nucleotides of each Pit-1 DNA response element were mutated in the rPRL-3kb gene promoter construct to disrupt Pit-1 DNA binding activity (Fig. 6AGo). Activin significantly inhibited the 3-kb parental promoter construct as well as the mutant promoter lacking the first Pit-1 site (Fig. 6BGo). However, removal of either site 2 or site 3 completely reversed the activin-mediated inhibition of the PRL gene (Fig. 6BGo). These results indicate that the Pit-1 sites 2 and 3, but not site 1, are important for activin-mediated repression of the PRL gene promoter activity.



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  		Fig. 6. Pit-1 Response Elements 2 and 3 in the Proximal Region of the PRL Promoter Are Critical for the Activin-Mediated Repression of PRL

A, Schematic representation of the rPRL-lux 3kb reporter construct. The proximal region contains three Pit-1 DNA-binding sites. For each of these sites, four nucleotides were changed to other bases as indicated to disrupt Pit-1 DNA binding. B, GH4C1 cells were transfected with either the wild-type rPRL-lux 3kb, Mut 1P, Mut 2P, or Mut 3P reporter constructs and the ß-galactosidase expression vector. Cells were stimulated or not with activin for 18 h, and luciferase activity was assessed. *, P < 0.05 compared with no activin treatment.

 
Activin Inhibits Pituitary Cell Growth through Menin
Activin is known to induce growth inhibition of epithelial, endothelial, lymphoid, and hematopoietic cells. Therefore, to next investigate the effect of activin on pituitary cell growth, GH4C1 cells were stimulated or not with activin or TGFß as a control. As shown in Fig. 7AGo, both activin and TGFß significantly inhibited cell growth of GH4C1 cells by 30%, thus indicating that activin acts as a potent cell growth inhibitor of lactotrope cells. To explore the function of menin in activin signaling, GH4C1 cells stably overexpressing antisense menin cDNA (GH4-AS), in which menin expression levels are strongly reduced (34), or control GH4C1 cells expressing the vector alone (GH4-V) were treated or not with activin or TGFß. Interestingly, both the activin and TGFß effect on cell growth inhibition were totally blocked in the absence of menin (Fig. 7AGo). To confirm these results, we used other means of blocking menin expression by utilizing modified phosphorothioate antisense oligonucleotides to menin. As shown in Fig. 7BGo, the effects of activin and TGFß on GH4C1 cell growth arrest were specifically antagonized and completely reversed in the presence of menin AS-oligo whereas they were not affected by the control oligonucleotide. These data demonstrate that activin acts as a potent pituitary tumor cell growth inhibitor and that this effect requires the tumor suppressor, menin.



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  		Fig. 7. Activin Inhibits Pituitary Cell Growth through the MEN1 Gene Product, Menin

A, GH4C1, GH4-V, and GH4-AS cells were cultured with or without TGFß or activin for 72 h, and cell viability was assessed by MTT assay. B, GH4C1 cells were cultured in the presence or the absence of menin antisense oligo or a control oligo and stimulated with or without TGFß or activin for 72 h. Cell viability was measured by MTT assay. *, P < 0.05 compared with no activin treatment (all panels).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Members of the activin/TGFß family of growth factors are important in regulating cell growth and production of a variety of hormones from different pituitary cell types. Our study defines a critical role for activin in regulating the growth and function of pituitary lactotrope cells. We demonstrate for the first time the requirement of the Smad pathway and the tumor suppressor menin in transducing activin signals to inhibition of PRL expression and cell growth arrest.

Our results also indicate that activin-mediated effects on PRL production are pituitary specific. PRL transcription is controlled through a series of distal and proximal enhancer elements that contain several DNA binding sites for the transcription factor Pit-1 that are required to regulate cell-specific expression (47). Our results clearly indicate that activin exerts a direct negative effect on Pit-1 gene expression. Thus, inhibition of Pit-1 expression by activin contributes to the decreased PRL level. Our data strongly support previous observations showing that extinction of PRL and GH gene expression in somatic cell hybrids is correlated with the repression of Pit-1 (48, 49). Interestingly, previous work showed that activin could contribute to reduced intracellular Pit-1 levels by decreasing its stability (31). Together with our results, this indicates that activin is able to down-regulate intracellular Pit-1 and PRL levels through different parallel mechanisms, highlighting the critical role played by activin in regulating pituitary hormone levels.

A previous study in GH3 pituitary cells found no apparent change in Pit-1 mRNA levels after activin stimulation of the cells for 24 h (30). Based on our study, activin-mediated repression of Pit-1 mRNA is a transient event that occurs within hours of stimulation and returns to normal by 24 h, thus explaining why Tamura et al. (30) failed to observe any change in Pit-1 mRNA in activin-treated cells.

Moreover, the inhibitory effect of activin on GH production is mediated through rapid phosphorylation of Pit-1, resulting in loss of DNA binding activity and protein instability (31). Together with our results, this suggests that activin acts at multiple levels to regulate Pit-1 and hormonal levels in pituitary cells.

We also show that the proximal, but not the distal, region of the PRL gene promoter mediates the activin-inhibitory effects. This proximal region of the PRL gene promoter contains three Pit-1 response elements, and we show here that sites 2 and 3 are each required for activin to repress PRL gene expression. Therefore, this suggests that activin-mediated inhibition of PRL expression is not only mediated through a direct down-regulation of Pit-1 gene expression but also through prevention of DNA binding of the preexisting pool of Pit-1 on sites 2 and 3 of the PRL gene promoter. Such interference with DNA binding and activating functions of Pit-1 has been described as a mechanism by which glucocorticoids inhibit PRL expression (46). Interestingly, negative regulation of PRL expression by glucocorticoids involves the same proximal Pit-1 sites 2 and 3, suggesting that this may represent a general mechanism of negative regulation of PRL gene transcription.

It is also possible that activin induces recruitment of a corepressor to the Pit-1 complex bound to DNA. Activin and TGFß are known to recruit histone deacetylases to some of their target genes, inhibiting their transcription (50). Pit-1 has also been shown to mediate transcriptional activation and repression by recruitment of coactivators or corepressors (51, 52). It will therefore be interesting in future studies to determine whether the Smads physically interact with Pit-1 and/or histone deacetylase activity can be found in these complexes.

Whereas Smads, menin and Pit-1, are critical to activin inhibition of PRL gene expression, a previous study suggested that the inhibitory effect of TGFß on the rat PRL gene promoter is Pit-1-independent (53). Therefore, our results also highlight significant differences in the intracellular signaling pathways of these two growth factors leading to PRL gene regulation in pituitary cells.

Inactivating alterations in the activin/TGFß signaling pathways have been described to form the basis of many human cancers (54). For instance, Smad2 and Smad4 are frequently mutated in colorectal cancers and pancreatic carcinomas, respectively (54). Truncated activin receptor forms are often found in human pituitary adenomas and function as dominant negative receptors, contributing to pituitary tumorigenesis by blocking the growth-inhibitory effect of activin (17). MEN1 is an autosomal dominant disorder characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas (32). Pituitary tumors occur in 30% of MENI patients, with prolactinomas being the most common type (56). Twenty-six percent of mice heterozygous for deletion of the MEN1 gene develop large pituitary tumors by 16 months of age (57). Here, we demonstrate that inactivation of menin through three different antisense technologies (cDNA antisense, oligonucleotide antisense, and siRNA) blocks activin signaling. This results in an increase in PRL gene expression, Pit-1 gene expression, and the loss of pituitary cell growth inhibition by activin. This is consistent with our previous work showing that inactivation of menin interrupts Smad3 binding to DNA, thereby blocking TGFß signaling (34). Thus, menin appears as a novel activin downstream signaling effector molecule, and its inactivation leads to the loss of activin/TGFß responses, emphasizing the critical role played by these growth factors in maintaining cellular homeostasis.

Pituitary adenoma is the most frequent adult intracranial neoplasm, accounting for 10% of brain tumors, and, in contrast to other neoplasms, occurs in younger patients. Prolactinomas are the most common hormone-secreting pituitary tumor, accounting for two-thirds of the patients in a pituitary tumor registry across the fourth decade (58). Prolactinomas often develop sporadically as a monoclonal proliferation, but the molecular mechanisms underlying the formation of these tumors remain largely unknown. Gene expression of Pit-1 has been explored in pituitary adenomas but revealed no mutation of the Pit-1 gene in these tumors, despite an up to 5-fold higher level of Pit-1 as compared with normal (59). To date, standard primary treatment using dopamine agonists showed suppressive, but not tumoricidal, effects with side effects and varying remission rates (60, 61). As for patients with therapeutic intolerance or insensitivity to dopamine agonists, surgery remains the last resort. Our study sheds light on the mechanisms by which activin regulates PRL, Pit-1 levels, and cell growth arrest in lactotrope cells, through the Smad pathway and the tumor suppressor menin, and opens new avenues for future therapies to combat human pituitary adenomas.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
GH4C1 cells, T47D, and CHO cells were cultured in DMEM (HyClone Laboratories, Inc., Logan, UT) in the presence of 10% fetal bovine serum (FBS) (HyClone), and 2 mM L-glutamine.

Cell Viability Assay [3-(4,5-Dimethylthiazolyl-2)-2,5 Diphenyltetrazoliumbromide (MTT)]
Cells were plated in triplicate at 5 x 103 cells per 100 µl in DMEM containing 2% FBS and cultured for 3 d in the presence or absence of 0.5 nM activin or 0.2 nM TGFß (PeproTech, Rocky Hill, NJ). Cell viability was assessed using the nonradioactive MTT cell growth assay for eukaryotic cells (Cell Titer 96, Promega G4000; Promega Corp., Madison, WI). Absorbance was measured at 570 nm with a reference wavelength at 450 nm using a Bio-Tek Microplate reader (Bio-Tek Instruments, Inc., Winooski, VT).

Transfection and Reporter Assays
GH4C1 cells were stably transfected with antisense menin cDNA as described (34). For luciferase assays, 0.3 µg of the different promoter constructs (rat PRL-lux 3kb, rat PRL-lux 450, rat PRL-lux mutants, human PRL-lux 5kb, human PRL-lux 250, and Pit-1-lux) were cotransfected in 106 cells with Lipofectamine (Invitrogen, Carlsbad, CA) with 0.3 µg of the pCH110 expression vector encoding ß-galactosidase, in the presence or absence of various Smad expression plasmids, as described in the legend of Fig. 2Go. Cells were trypsinized 1 d after transfection, divided in three, allowed to recover, and then serum-starved with or without activin (0.5 nM) or TGFß (0.2 nM) for 18 h. Cells were then washed with PBS and lysed in 100 µl lysis buffer (1% Triton X-100; 15 mM MgSO4; 4 mM EGTA; 1 mM dithiothreitol; 25 mM glycylglycine, pH 7.8) on ice. The luciferase activity of each sample was measured using 45 µl cell lysate (luminometer from EG&G Berthold, Badwildbad, Germany) and normalized to the relative ß-galactosidase activity. CHO cells were transfected using the calcium phosphate method. Briefly, 1 µg of the different reporter constructs were transfected with 1 µg ß-galactosidase expression vector. Cells were serum-starved 1 d after transfection, in the presence or absence of 0.5 nM activin, for 18 h. Luciferase assays were performed as for GH4C1 cells.

Western Blot Analysis
For long time courses (0–24 h), GH4C1 and T47D cells were plated at 106 cells/ml in DMEM containing 2% FBS and stimulated in the presence or absence of 0.5 nM activin. For short time courses (0–60 min), cells were serum starved overnight, and stimulated or not with 0.5 nM activin. Cells were lysed on ice in lysis buffer [50 mM HEPES at pH 7.5, 150 mM sodium chloride, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 5 mM EDTA, 10% glycerol, 0.5% Nonidet P40, and 0.5% sodium deoxycholate] supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 µg/ml pepstatin. Total cell extracts were then separated on polyacrylamide gels, transferred to nitrocellulose, and incubated with the indicated specific antibody overnight at 4 C [menin (62), stat3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), PRL, phospho-Smad2 (Upstate Biotechnology, Inc., Lake Placid, NY), Smad2/3 (Santa Cruz), tubulin (Sigma Chemical Co., St. Louis, MO), Pit-1 (Santa Cruz)]. After incubation, membranes were washed twice for 10 min in washing buffer (50 mM Tris-Cl at pH 7.6, 200 mM NaCl, 0.05% Tween 20) and incubated with a secondary antibody coupled to horseradish peroxidase (Sigma) at 1:10,000 dilution) for 1 h at room temperature. Then, membranes were washed four times for 15 min in washing buffer. Immunoreactivity was normalized by chemiluminescence (Lumi-Light Plus Western blotting substrate, Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions and revealed using an Alpha Innotech Fluorochem Imaging system (Packard Canberra, Montreal, Quebec, Canada). Densitometric analysis was performed using Fluorochem 8000 software (Alpha Innotech, San Leandro, CA) that allows normalization and quantitative analysis of chemiluminescence under nonsaturating condition.

Northern Blot Analysis
GH4C1 (106 cells/ml) were plated in DMEM containing 2% FBS and stimulated with 0.5 nM activin for different periods of time. Total RNA was extracted using Trizol (Invitrogen). Each sample (20 µg) was then separated on agarose gels (1% agarose in 0.04 M 3-(N-morpholino)propanesulfonic acid; 0.01M sodium acetate; 10 mM EDTA, pH 8.0; and 2.5M formaldehyde) and transferred to nylon membranes. Membranes were rinsed twice in 10x standard saline citrate, cross-linked under UV light, and prehybridized in a prehybridization solution (0.5M NaPO4, pH 7.2; 1 mM EDTA, pH 8.0; 7% sodium dodecyl sulfate (SDS); 1% BSA; and 200 µg/ml salmon sperm DNA) for 2 h at 60 C. Probes for PRL, Pit-1, and GAPDH were labeled using the Random Priming Kit (Roche), and then add to the prehybridization solution for an overnight incubation. Membranes were washed twice for 15 min with wash A (40 mM NaPO4, pH 7.2; 5% SDS; 1 mM EDTA; and 0.5% BSA), and four times with wash B (40 mM NaPO4, pH 7.2; 1% SDS; 1 mM EDTA). Results were revealed using a phosphor imager Cyclone Storage Phosphor Screen (Packard Instruments, Meriden, CT).

Antisense Oligonucleotide (AS-Oligo) Treatment
Phosphorothioate-derivatized antisense menin and control oligonucleotides (C-oligos) (20 bp) were synthesized. The AS-oligo was 5'-GGGCGGCCTTCAGCCCCATG-3', and the C-oligo was 5'-TCAGACTGGCTCTCTCCATG-3'. Cells were plated in the presence or absence of 100 µM menin AS-oligo or control C-oligo for 12 h and then stimulated with 0.5 nM activin or 0.2 nM TGFß. Luciferase activity was measured after 18 h and cell growth was measured after 72 h of ligand stimulation.

Mutagenesis of the rPRL Promoter
The three Pit-1 proximal binding sites in the wild-type PRL-3kb construct were mutated singly to disrupt DNA binding of Pit-1 using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotides used for mutagenesis were: mut-1P, 5'-GCCTGATTATATATATGGGAATGAAGGTGTCGAAGG-3' and 5'-CCTTCGACACCTTCATTCCCATATATATAATCAGGC-3'; mut-2P, 5'-GGCCACTATGTCTTCCTGAATATTCCGAAGAAATAAAATACCATTTGA-3' and 5'-TCAAATGGTATTTTATTTCTTCGGAATATTCAGGAAGACATAGTGGCC-3'; mut-3P, 5'-TCATTTCCTTTTGCTGTAATTGCGAAAAATCCTTCCTTTCTGGCC-3' and 5'-GGCCAGAAAGGAAGGATTTTTCGAAATTACAGCAAAAGGAAATGA-3'. The mutated nucleotides are indicated in bold. All mutant constructs were confirmed by sequencing.

siRNA Design, Generation, and Transfection
Three different siRNAs corresponding to distinct parts of the rat MEN1 gene (GenBank accession no. 9506894) were designed using the siRNA Selection Program (Whitehead Institute for Biomedical Research, Cambridge, MA). All siRNA sequences were Blast searched in the National Center for Biotechnology Information search for short-nearly exact-matches mode against all rat sequences of the GenBank database and were not found to have significant homology to genes other than the rat MEN1 gene. siRNAs were synthetized using the Silencer siRNA Construction Kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. SiRNAs sequences were as follows: M10 5'-AAGGCCGCCCAGAAGACGCTG-3'; M379 5'-AACAGCCTCAGCCGCTCCTAC-3'; M1073 5'-AAGAGATCTACAAGGAATTCT-3'. GAPDH siRNA was provided with the kit. GH4C1 cells (5 x 104) were transfected using the siPORT Lipid transfection agent (Ambion) according to the manufacturer’s instructions. Expression levels of menin, PRL, and Pit-1 were analyzed by Western blot and RT-PCR.

Statistical Analysis
Results are expressed as mean ± SD. Differences were assessed by one-way ANOVA or the unpaired t test, where appropriate. P < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. Eto and Ajinomoto Co., Inc. for activin; Dr. J. Massagué for 3TPLux; Drs. J. Wrana and L. Attisano for {Delta}NSmad2; Dr. H. Lodish for {Delta}NSmad3; and Dr. H. Kaji for GH4-V and GH4-AS cells.


   FOOTNOTES
 
This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP-53141 (to J.J.L.), Grant MOP-9315 (to G.N.H.), and Grant MOP-13681 (to S.A.). S.A. and J.J.L. are Research Scientists of the Canadian Cancer Society through an Award from the National Cancer Institute of Canada (NCIC). A.L. is a recipient of a studentship from the Fonds de la recherche en santé du Quebec (FRSQ), L.C. is a recipient of a studentship from the NCIC.

E.L. and E.H.L. contributed equally to this work and should both be considered first authors.

Abbreviations: AS-oligo, menin antisense oligonucleotide; CHO, Chinese hamster ovary; C-oligo, control oligonucleotide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human; MEN1, multiple endocrine neoplasia type 1, MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide; PRL, prolactin; r, rat; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; Stat, signal transducer and activator of transcription.

Received for publication December 5, 2003. Accepted for publication March 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Luisi S, Florio P, Reis F, Petraglia F 2001 Expression and secretion of activin A: possible physiological and clinical implications. Eur J Endocrinol 145:225–236[CrossRef][Medline]
  2. Lebrun JJ, Chen Y, Vale WW 1997 Receptor serine kinases and signaling by activins and inhibins. In: Vale WW, ed. Inhibin, activin and follistatin: regulatory functions in system and cell biology. New York: Springer-Verlag; 1–21
  3. Murata T, Ying S 1991 Transforming growth factor-ß and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc Soc Exp Biol Med 198:599–605[Abstract]
  4. Lebrun JJ, Vale WW 1997 Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation. Mol Cell Biol 17:1682–1691[Abstract]
  5. McCarthy SA, Bicknell R 1993 Inhibition of vascular endothelial cell growth by activin-A. J Biol Chem 268:23066–23071[Abstract/Free Full Text]
  6. Brosh N, Sternberg D, Honigwachs-Sha’anani J, Lee BC, Shav-Tal Y, Tzehoval E, Shulman LM, Toledo J, Hacham Y, Carmi P, Jiang W, Sasse J, Horn F, Burstein Y, Zipori D 1995 The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A. J Biol Chem 270:29594–29600[Abstract/Free Full Text]
  7. Valderrama-Carvajal H, Cocolakis E, Lacerte A, Lee EH, Krystal G, Ali S, Lebrun JJ 2002 Activin/TGF-ß induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat Cell Biol 4:963–969[CrossRef][Medline]
  8. Kalkhoven E, Roelen BA, de Winter JP, Mummery CL, van den Eijnden-van Raaij AJ, van der Saag PT, van der Burg B 1995 Resistance to transforming growth factor ß and activin due to reduced receptor expression in human breast tumor cell lines. Cell Growth Differ 6:1151–1161[Abstract]
  9. Liu QY, Niranjan B, Gomes P, Gomm JJ, Davies D, Coombes RC, Buluwela L 1996 Inhibitory effects of activin on the growth and morphogenesis of primary and transformed mammary epithelial cells. Cancer Res 56:1155–1163[Abstract/Free Full Text]
  10. Cocolakis E, Lemay S, Ali S, Lebrun JJ 2001 The p38 MAPK pathway is required for cell growth inhibition of human breast cancer cells in response to activin. J Biol Chem 276:18430–18436[Abstract/Free Full Text]
  11. Yasuda H, Mine T, Shibata H, Eto Y, Hasegawa Y, Takeuchi T, Asano S, Kojima I 1993 Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. J Clin Invest 92:1491–1496
  12. Xu J, McKeehan K, Matsuzaki K, McKeehan WL 1995 Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism. J Biol Chem 270:6308–6313[Abstract/Free Full Text]
  13. Zauberman A, Oren M, Zipori D 1997 Involvement of p21(WAF1/Cip1), CDK4 and Rb in activin A mediated signaling leading to hepatoma cell growth inhibition. Oncogene 15:1705–1711[CrossRef][Medline]
  14. Takabe K, Lebrun JJ, Nagashima Y, Ichikawa Y, Mitsuhashi M, Momiyama N, Ishikawa T, Shimada H, Vale WW 1999 Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell proliferation. Endocrinology 140:3125–3132[Abstract/Free Full Text]
  15. Chen W, Woodruff TK, Mayo KE 2000 Activin A-induced HepG2 liver cell apoptosis: involvement of activin receptors and smad proteins. Endocrinology 141:1263–1272[Abstract/Free Full Text]
  16. Su GH, Bansal R, Murphy KM, Montgomery E, Yeo CJ, Hruban RH, Kem SE 2001 ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma. Proc Natl Acad Sci USA 98:3254–3257[Abstract/Free Full Text]
  17. Zhou Y, Sun H, Danila DC, Johnson SR, Sigai DP, Zhang X, Klibanski A 2000 Truncated activin type I receptor Alk4 isoforms are dominant negative receptors inhibiting activin signaling. Mol Endocrinol 14:2066–2075[Abstract/Free Full Text]
  18. Attisano L, Wrana JL, Montalvo E, Massague J 1996 Activation of signalling by the activin receptor complex. Mol Cell Biol 16:1066–1073[Abstract]
  19. Willis SA, Zimmerman CM, Li LI, Mathews LS 1996 Formation and activation by phosphorylation of activin receptor complexes. Mol Endocrinol 10:367–379[Abstract]
  20. Abdollah S, Macias-Silva M, Tsukazaki T, Hayashi H, Attisano L, Wrana JL 1997 TbRI phosphorylation of Smad2 of Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem 272:27678–27685[Abstract/Free Full Text]
  21. Lebrun J-J, Takabe K, Chen Y, Vale W 1999 Roles of pathway-specific and inhibitory Smads in activin receptor signaling. Mol Endocrinol 13:15–23[Abstract/Free Full Text]
  22. Lagna G, Hata A, Hemmati-Brivanlou A, Massague J 1996 Partnership between DPC4 and SMAD proteins in TGF-ß signalling pathways. Nature 383:832–836[CrossRef][Medline]
  23. Macias-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, Wrana JL 1996 MADR2 is a substrate of the TGFß receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87:1215–1224[CrossRef][Medline]
  24. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P 1997 TGF-b receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16:5353–5362[CrossRef][Medline]
  25. Derynck R, Zhang Y, Feng XH 1998 Smads: transcriptional activators of TGF-ß responses. Cell 95:737–740[CrossRef][Medline]
  26. Pangas SA, Woodruff TK 2000 Activin signal transduction pathways. Trends Endocrinol Metab 11:309–314[CrossRef][Medline]
  27. Bodner M, Castrillo J, Theill L, Deerinck T, Ellisman M, Karin M 1988 The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 55:505–518[CrossRef][Medline]
  28. Ingraham H, Chen R, Mangalam H, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519–529[CrossRef][Medline]
  29. Li S, Crenshaw IE, Rawson E, Simmons D, Swanson L, Rosenfeld M 1990 Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene Pit-1. Nature 347:528–533[CrossRef][Medline]
  30. Tamura N, Irahara M, Kuwahara A, Ushigoe K, Sugino H, Aono T 2000 Effect of activin on production and secretion of prolactin and growth hormone in cultured rat GH3 cells. Eur J Endocrinol 142:506–511[Abstract]
  31. Gaddy-Kurten D, Vale WW 1995 Activin increases phosphorylation and decreases stability of the transcription factor Pit-1 in MtTW15 somatotrope cells. J Biol Chem 270:28733–28739[Abstract/Free Full Text]
  32. Guo S, Sawicki M 2001 Molecular and genetic mechanisms of tumorigenesis in multiple endocrine neoplasia type-1. Mol Endocrinol 15:1653–1664[Abstract/Free Full Text]
  33. Namihira H, Sato M, Murao K, Cao WM, Matsubara S, Imachi H, Niimi M, Dobashi H, Wong NC, Ishida T 2002 The multiple endocrine neoplasia type 1 gene product, menin, inhibits the human prolactin promoter activity. J Mol Endocrinol 29:297–304[Abstract]
  34. Kaji H, Canaff L, Lebrun J-J, Goltzman D, Hendy GN 2001 Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type b signaling. Proc Natl Acad Sci USA 98:3837–3842[Abstract/Free Full Text]
  35. Keech C, Gutierrez-Hartmann A 1989 Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3', 5'-cyclic adenosine monophosphate-regulated gene expression in vivo. Mol Endocrinol 3:832–839[Abstract]
  36. Keech C, Gutierrez-Hartmann A 1991 Insulin activation of rat prolactin promoter activity. Mol Cell Endocrinol 78:55–60[CrossRef][Medline]
  37. Tashjian Jr AH, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352[Medline]
  38. Song JY, Jin L, Lloyd RV 1989 Effects of estradiol on prolactin and growth hormone messenger RNAs in cultured normal and neoplastic (MtT/W15 and GH3) rat pituitary cells. Cancer Res 49:1247–1253[Abstract/Free Full Text]
  39. Nelson C, Albert VR, Elsholtz H, Lu LI-W, Rosenfeld M 1988 Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239:1400–1405[Abstract/Free Full Text]
  40. Berwaer M, Monget P, Peers B, Mathy-Hartert M, Bellefroid E, Davis JR, Belayew A, Martial JA 1991 Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence. Mol Cell Endocrinol 80:53–64[CrossRef][Medline]
  41. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone Jr MA, Wrana JL, Falb D 1997 The MAD-related protein Smad7 associates with the TGFß receptor and functions as an antagonist of TGFß signaling. Cell 89:1165–1173[CrossRef][Medline]
  42. Liu X, Sun Y, Constantinescu SN, Karam E, Weinberg RA, Lodish HF 1997 Transforming growth factor ß-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci USA 94:10669–10674[Abstract/Free Full Text]
  43. Ginsburg E, Vonderhaar BK 1995 Prolactin synthesis and secretion by human breast cancer cells. Cancer Res 55:2591–2595[Abstract/Free Full Text]
  44. Ramamoorthy P, Sticca R, Wagner TE, Chen WY 2001 In vitro studies of a prolactin antagonist, hPRL-G129R in human breast cancer cells. Int J Oncol 18:25–32[Medline]
  45. Chen RP, Ingraham H, Treacy MN, Albert VR, Wilson L, Rosenfeld M 1990 Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements. Nature 346:583–586[CrossRef][Medline]
  46. Nalda AM, Martial JA, Muller M 1997 The glucocorticoid receptor inhibits the human prolactin gene expression by interference with Pit-1 activity. Mol Cell Endocrinol 134:129–137[CrossRef][Medline]
  47. Crenshaw IE, Kalla K, Simmons D, Swanson L, Rosenfeld M 1989 Cell-specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between promoter and enhancer elements. Genes Dev 3:959–972[Abstract/Free Full Text]
  48. MacComick A, Wu D, Castrillo J, Dana S, Strobl J, Thompson EB, Karin M 1988 Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific trans-activator GHF-1. Cell 55:379–389[CrossRef][Medline]
  49. Supowit SC, Ramsey T, Thompson EB 1992 Extinction of prolactin gene expression in somatic cell hybrids is correlated with the repression of the pituitary-specific trans-activator GHF-1/Pit-1. Mol Endocrinol 6:786–792[Abstract]
  50. Wotton D, Lo RS, Swaby LA, Massague J 1999 Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem 274:37105–37110[Abstract/Free Full Text]
  51. Scully KM, Jacobson EM, Jepsen K, Lunyak V, Viadiu H, Carriere C, Rose DW, Hooshmand F, Aggarwal AK, Rosenfeld MG 2000 Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290:1127–1131[Abstract/Free Full Text]
  52. Xu L, Lavinsky RM, Dasen JS, Flynn SE, McInerney EM, Mullen TM, Heinzel T, Szeto D, Korzus E, Kurokawa R, Aggarwal AK, Rose DW, Glass CK, Rosenfeld MG 1998 Signal-specific co-activator domain requirements for Pit-1 activation. Nature 395:301–306[CrossRef][Medline]
  53. Farrow KN, Gutierrez-Hartmann A 1999 Transforming growth factor-ß1 inhibits rat prolactin promoter activity in GH4 neuroendocrine cells. DNA Cell Biol 18:863–873[CrossRef][Medline]
  54. Gold LI 1999 The role for transforming growth factor-ß (TGF-ß) in human cancer. Crit Rev Oncog 10:303–360[Medline]
  55. Deleted in proof
  56. Burgess J, Shepherd J, Parameswaran V, Hoffman L, Greenaway T 1996 Prolactinomas in a large kindred with multiple endocrine neoplasia type 1: clinical features and inheritance pattern. J Clin Endocrinol Metab 81:1841–1845[Abstract]
  57. Crabtree JS, Scacheri PC, Ward JM, Garrett-Beal L, Emmert-Buck MR, Edgemon KA, Lorang D, Libutti SK, Chrandrasekharappa SC, Marx SJ, Spiegel AM, Collins FS 2001 A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc Natl Acad Sci USA 98:1118–1123[Abstract/Free Full Text]
  58. Drange MR, Fram NR, Herman-Bonert V, Melmed S 2000 Pituitary tumor registry: a novel clinical resource. J Clin Endocrinol Metab 85:168–174[Abstract/Free Full Text]
  59. Pellegrini I, Barlier A, Gunz G, Figarella-Branger D, Enjalbert A, Grisoli F, Jaquet P 1994 Pit-1 gene expression in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 79:189–196[Abstract]
  60. Colao A, Merola B, Sarnacchiaro F, Di Sarno A, Landi ML, Marzullo P, Cerbone G, Ferone D, Lombardi G 1995 Comparison among different dopamine-agonists of new formulation in the clinical management of macroprolactinomas. Horm Res 44:222–228[Medline]
  61. Molitch ME, Thorner MO, Wilson C 1997 Management of prolactinomas. J Clin Endocrinol Metab 82:996–1000[Free Full Text]
  62. Kaji H, Canaff L, Goltzman D, Hendy GN 1999 Cell cycle regulation of menin expression. Cancer Res 59:5097–5101[Abstract/Free Full Text]



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