help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Castillo, A. I.
Right arrow Articles by Aranda, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Castillo, A. I.
Right arrow Articles by Aranda, A.
Endocrinology Vol. 138, No. 12 5442-5451
Copyright © 1997 by The Endocrine Society

ARTICLES

Differential Regulation of Pituitary-Specific Gene Expression by Insulin-Like Growth Factor 1 in Rat Pituitary GH4C1 and GH3 Cells1

Ana Isabel Castillo and Ana Aranda

Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas, Madrid 28029, Spain

Address all correspondence and requests for reprints to: Ana Aranda, Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: Aaranda{at}.iib.uam.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have compared the influence of insulin-like growth factor 1 (IGF-1) on pituitary gene expression in the rat cell lines GH4C1 and GH3. Incubation with IGF-1 increased PRL messenger RNA (mRNA) levels in GH4C1 cells by 4- to 5-fold but decreased the levels of PRL transcripts in GH3 cells. In addition, the levels of GH-mRNA that were not affected by IGF-1 in GH4C1 cells were significantly inhibited by the growth factor in GH3 cells. IGF-1 also decreased PRL and GH-mRNA response to T3, retinoic acid, and Fk in GH3 cells. Stability of PRL or GH transcripts was not altered by IGF-1 in GH3 cells, suggesting that the inhibitory effect is exerted at a transcriptional level. The pituitary-specific transcription factor GHF-1/Pit-1 activates both the GH and PRL promoters. As analyzed by Western blot, IGF-1 did not alter GHF-1/Pit-1 protein levels in GH4C1 cells but reduced the levels of the transcription factor in GH3 cells. This decrease is secondary to a reduction of GHF-1/Pit-1 transcripts in IGF-1-treated GH3 cells. Thus, a different effect of IGF-1 on the expression of GHF-1/Pit-1 in GH3 and GH4C1 cells is likely involved in the different regulation of GH and PRL gene in both cell types. IGF-1 increases the activity of the PRL promoter in transient transfection assays in GH4C1 cells by a Ras-dependent mechanism. Expression of oncogenic RasVal12 mimics the effect of IGF-1, and the dominant negative RasAsn17 blocks IGF-1-mediated stimulation of the PRL promoter in GH4C1 cells. Although IGF-1 did not stimulate the PRL promoter in GH3 cells, RasVal12 strongly activated the promoter in these cells. Hence, the machinery to activate Ras-dependent signaling is intact in GH3 cells. Moreover, IGF-1 stimulates the mitogen-activated protein kinase in GH3 cells, showing that the components linking the IGF-1 receptor to Ras are also active. These results suggest that, in addition to the Ras/mitogen-activated protein kinase pathway, IGF-1 could activate a different pathway and that the combination of both is required to elicit PRL gene expression by the growth factor. This second pathway may be defective in GH3 cells that respond to Ras but not to IGF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RAT tumor cell lines have been widely used for studies of regulation of expression of pituitary cells, because they retain the capacity to synthesize and secrete GH and PRL in a hormone-regulated manner. Expression of both genes is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restricted manner in cells of the anterior pituitary (1, 2). Multiple hormones, growth factors, and oncogenes act in conjunction with GHF-1/Pit-1 to regulate pituitary-specific expression of the PRL and GH genes.

The actions of insulin-like growth factor 1 (IGF-1) are mediated by ligand activation of a tyrosine kinase receptor. IGF-1 receptors are expressed in a variety of cell types, including anterior pituitary cells and pituitary cell lines (3). Binding of IGF-1 is followed by a rapid activation of the intrinsic tyrosine activity of the receptor, phosphorylation of several protein substrates, and activation (among others) of phosphatidylinositol-3 kinase and Ras. Activation of Ras initiates an intracellular signaling cascade of serine/threonine kinases. Raf, MEK, and mitogen-activated protein (MAP) kinases are downstream components of this pathway, which has been shown to result in phosphorylation of numerous substrates, including nuclear transcription factors (4, 5). A rapid and strong activation of the MAP kinases (MAP-K), ERK-1 and ERK-2, by IGF-1 recently has been observed in the rat pituitary GC cell line (6).

It has been described that IGF-1 suppresses basal GH-messenger RNA (GH-mRNA) levels, as well as GH-releasing hormone (GH-RH)- and T3-induced GH-mRNA stimulation (7) in primary cultures of rat pituitary cells. This suppression has been shown to be secondary to a direct inhibition of the GH gene transcription rate (8, 9), although the relationship, if any, between activation of the Ras/MAP-K pathway and IGF-1-mediated suppression of GH gene transcription remains to be established. The inhibition of GH gene transcription by IGF-1 in primary pituitary cells is selective, because expression of the PRL gene was unaffected by IGF-1 in these cells (7). In contrast with the results obtained in primary cultures, we very recently have observed that GH-mRNA levels are not suppressed by IGF-1 in GH4C1 cells, a rat pituitary tumor cell line. Furthermore, the growth factor significantly induces PRL gene expression in GH4C1 cells through sequences located in the proximal promoter (Castillo, A. I., R. Tolon, and A. Aranda, manuscript submitted). It has been shown that expression of a constitutively active Ras oncogene produces a strong activation of the PRL promoter and that Raf, MAP-K, and Ets-1 are crucial components of the downstream transmission of the Ras signal in the regulation of PRL promoter activity (11, 12, 13). Our results have demonstrated that activation of the PRL promoter by IGF-1 in GH4C1 cells requires activation of the Ras/MAP-K pathway, because dominant negative mutants of these proteins block stimulation of PRL gene expression by IGF-1. Our results also suggest that IGF-1 could stimulate additional, independent pathway(s), which cooperate with the Ras/MAP-K to elicit PRL gene expression.

In this study, we have compared the regulation of PRL, GH, and GHF-1/Pit-1 gene expression in pituitary GH4C1 and GH3 cells. Both cell lines are closely related and have retained the ability of normal somatolactototropes to produce GH and PRL. The GH3 cells were cloned from pituitary tumor cells transferred twice between animal and tissue culture, and the GH4C1 cells are a cloned variant of the GH3 cells (14). Our results show a strikingly different regulation of these genes by IGF-1. In GH4C1 cells, the growth factor induces PRL gene expression without significantly affecting the GH gene. In contrast, IGF-1 strongly inhibits PRL and GH gene expression in GH3 cells. This occurs despite the fact that oncogenic Ras activates the PRL promoter in GH3 cells and that IGF-1 activates the MAP-Ks in a manner indistinguishable from that found in GH4C1 cells. A differential regulation of GHF-1/Pit-1 might contribute to the different regulation of GH and PRL gene expression in both cell lines. Expression of the GHF-1/Pit-1 gene is suppressed by IGF-1 in GH3 cells, where the growth factor inhibits pituitary-specific gene expression, but is not reduced in GH4C1 cells, where it activates the PRL gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA extraction and hybridization
GH4C1 cells were grown in DMEM containing 10% FCS, and GH3 cells were cultured in RPMI medium containing 15% donor horse serum and 2.5% FCS (GIBCO BRL, Grand Island, NY). The cells were incubated for 24 h in a medium containing 10% AG1-X8 resin-charcoal stripped newborn calf serum and then treated with IGF-1, insulin, triiodothyronine (T3), forskolin (Fk), or retinoic acid (RA) in serum-free medium, as indicated in the figures. Total RNA was extracted from the cell cultures with guanidine thiocyanate (15). The RNA was run in 1% formaldehyde-agarose gels and transferred to nylon-nitrocellulose membranes (Nytran, Schleicher & Schuell, Kenne, NH) for Northern blot analysis. The RNA was stained with 0.02% methylene blue. The blots were hybridized with labeled cDNA probes for rat PRL (16), GH (17), or GHF-1 (18). Hybridizations were carried out at 421/4 C with 50% formamide and the more stringent wash was at 421/4 C with 1xSSC-0.1% SDS. Quantification of mRNA levels was carried out by densitometric scan of the autoradiograms. The values obtained were always corrected by the amount of RNA applied in each lane, which was determined by densitometry of the stained membranes.

Plasmids
GH-chloramphenicol acetyltransferase (CAT) constructs containing -530 bp of the rat GH promoter have been described previously (19). The plasmid -400rGHF-1-CAT (20) contains sequences of the rat GHF-1 promoter ligated to CAT. The plasmids -3000Prl-CAT and -176Prl-CAT contain 5'-flanking sequences of the rat PRL promoter (21). Constitutive expression vectors for oncogenic Ha-rasVal12 (22), the dominant inhibitory Ha-rasAsn17 mutant (23), GHF-1 (24), and the IGF-1 receptor (pBPV-IGF1-R) (25) also were used in transient transfection assays.

Cell culture and transfections
GH4C1 or GH3 cells were transfected by electroporation, as previously described (19, 26, 27). Ten micrograms of the reporter plasmids were mixed with 20–30 million cells and exposed to a high-voltage pulse (200–250 V, 960 µF) by using a Bio-Rad (Bio-Rad Laboratories, Richmond, CA) electroporator with a capacitor extender. The cells from each electroporation were split into different culture plates and incubated overnight in DMEM containing 10% AG1x8 resin-charcoal stripped newborn calf serum. This hormone-depleted medium was replaced by serum-free medium, and the treatments were administered. In some assays, in which the cells were cotransfected with the PRL reporter construct and expression vectors, the amount of DNA was kept constant by addition of the same amount of an empty noncoding vector (RSV-0). Each transfection also received 2.5 µg of a luciferase vector to monitor transfection efficiency. Each treatment with the ligands was performed, at least, in duplicate cultures that normally exhibited less than 10% variation in CAT activity. CAT activity was determined by incubation of the cell extracts with [14C]chloramphenicol. The unreacted and acetylated [14C]chloramphenicol were separated by TLC, identified by autoradiography, and quantified. The data are expressed as the percentage of acetylated forms after each treatment. Each experiment was repeated at least two or three times with similar relative differences in regulated expression.

Western blot analysis
The levels of GHF-1/Pit-1 were determined by Western blot analysis. GH4C1 or GH3 cell extracts (10 µg) were run in a 12% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane and incubated with a 1:1000 dilution of a polyclonal antibody that recognizes the transcription factor, and GHF-1/Pit-1 was identified by chemiluminescence (24).

Determination of MAP-K activity
GH4C1 and GH3 cells were plated in 60-mm diameter dishes in growth medium. After 24 h, cells were switched to a resin-charcoal depleted medium, and after an additional 12 h period, were serum starved for 18 h and exposed to 13 nM IGF-1 or 100 nM phorbol 12-myristate 13-acetate (TPA) for different time periods. Cells were then lysed in 300 µl of cold lysis buffer (10 mM EGTA, 40 mM ß-glycerophosphate, 1% NP-40, 2.5 mM MgCl2, 2 mM orthovanadate, 1 mM Dithiothreitol, 20 mM HEPES, pH 7, 5) containing protease inhibitors (leupeptin, aprotinin, benzamidine, and phenylmethylsulfonyl fluoride). Cell extracts (100 µg) were incubated with 5 µl ERK-2 (C-14) rat polyclonal antibody (Santa Cruz Biotechnology, Gebuhrenfrei, Germany) at 4 C for 1 h. Protein A-Sepharose was added and the samples incubated for an additional hour and centrifuged. The pellets were washed three times with PBS, 1% NP-40, and 2 mM orthovanadate; and once with 0.1 M Tris-ClH (pH 7.5), 0.5 M LiCl, and kinase buffer (2 mM Dithiothreitol, 20 mM glycerophosphate, 20 mM PnPP, 20 mM MgCl2, 0.1 mM orthovanadate, and 20 mM HEPES, pH 7.6). The immunoprecipitates were then incubated in 30 µl of kinase buffer containing 20 µM ATP, 2 µCi of {gamma}-32P-ATP, and 1 µg myelin basic protein (MBP) as a substrate. After incubation for 20 min at 30 C, the samples were subjected to SDS-PAGE in 15% acrylamide gels, dried, and autoradiographed. MBP band density on the autoradiographs was quantitated by densitometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influence of IGF-1 on PRL- and GH-mRNA levels in GH4C1 and GH3 cells
Figure 1Go compares the effect of a 48-h incubation with 13 nM IGF-1 on PRL and GH-mRNA levels in GH4C1 and GH3 cells. IGF-1 significantly increased (4- to 5-fold) PRL transcripts in GH4C1 cells. In contrast with these results, IGF-1 caused a strong decrease of PRL mRNA levels in GH3 cells. A reduction of GH-mRNA levels also was observed in IGF-1-treated GH3 cells, whereas this factor did not significantly alter GH transcripts in GH4C1 cells. Quantification of Northern blots from five different experiments showed that IGF-1 reduced by 40–75% Prl- and GH-mRNA levels in GH3 cells. In GH41 cells, GH-mRNA levels only varied between 0 and 15% after treatment with IGF-1.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. IGF-1 inhibits the effect of T3 on PRL and GH transcripts in GH3 cells. The cells were incubated with 13 nM IGF-1 and/or 5 nM T3 for 48 h. Northern blot analyses were carried out with 20 µg of total RNA from GH3 cells (right panels) and GH4C1 cells (left panels). The blots were sequentially hybridized with labeled cDNA probes for PRL (Prl) and GH. The lower panels show the ribosomal RNAs (rRNA), stained with methylene blue.

 
The effect of IGF-1 in combination with T3 on PRL and GH gene expression also was analyzed in both cell lines. Treatment with 5 nM T3 stimulated PRL mRNA levels in both GH4C1 and GH3 cells. In GH3 cells, this response was strongly suppressed in the presence of IGF-1, whereas IGF-1 did not inhibit this response in GH4C1 cells. T3 consistently induced GH gene expression in both cell lines, and IGF-1 was able to partially inhibit the stimulatory effect of T3 on GH gene expression in GH3 cells. In the experiment illustrated in the figure, induction by T3 decreased from 6-fold in the absence of IGF-1, to 4-fold in its presence. This inhibition was less marked in GH4C1 cells and was not consistently observed in independent experiments.

We also examined the response to IGF-1 in RA-treated cells. Figure 2Go shows GH and PRL-mRNA levels in GH3 and GH4C1, incubated for 48 h with 1 µM RA in the presence and absence of IGF-1. In results similar to those obtained with T3, IGF-1 reduced the GH response to RA in GH3 cells. In addition, we observed that RA increases PRL transcripts in both GH4C1 and GH3 cells. The induction of PRL-mRNA by RA is less marked than that of GH-mRNA, but it was consistently observed in different experiments. IGF-1 also decreased significantly the PRL response to RA in GH3 cells, whereas it acted additively with RA to increase PRL transcripts in GH4C1 cells.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 2. Influence of IGF-1 and RA on PRL and GH transcripts in pituitary GH4C1 and GH3 cells. Northern blot analysis was carried out with RNA extracted from cells incubated for 48 h with 13 nM IGF-1 alone or in combination with 1 µM RA, as indicated.

 
Figure 3Go shows the influence of incubation with IGF-1 on the PRL response to 10 µM Fk. Again, IGF-1 significantly reduced the basal levels of PRL transcripts in GH3 cells. Incubation with Fk for 48 h increased by about 2-fold PRL-mRNA and significantly attenuated the inhibitory response of IGF-1. In contrast, IGF-1 induced PRL-mRNA levels more strongly than Fk in GH4C1 cells, and these levels were maximal with the combination of Fk and IGF-1.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Fk antagonizes the inhibitory effect of IGF-1 in GH3 cells. GH-mRNA and PRL-mRNA levels were determined in cells incubated for 48 h with 13 nM IGF-1 and/or 10 µM Fk.

 
Regulation of PRL promoter activity in GH4C1 and GH3 cells
The 5'-flanking region of the PRL gene mediates transcriptional responses to several hormones and second messengers. Experiments, in which GH4C1 and GH3 cells are transfected with a Prl-CAT construct containing 3000 bp of the rat PRL promoter, are shown in Fig. 4AGo. Incubation with IGF-1 produced a significant increase of CAT activity in GH4C1 cells transfected with the -3000Prl-CAT construct. The proximal region of the PRL gene is sufficient to mediate this response, because IGF-1 increases the activity of a PRL promoter construct containing only 176 bp (-176Prl-CAT) to an extent similar to that found with -3000Prl-CAT (data not shown). Incubation with Fk activated the -3000Prl-CAT construct in GH4C1 cells (28, 29), and CAT activity was maximal in cells incubated with the combination of IGF-1 and Fk. However, Fk caused an apparent attenuation of the IGF-1 response. Fk treatment seems to decrease the IGF-1 response from 3-fold in the absence of the cAMP stimulator, to only 2-fold in its presence.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Comparison of the effect of IGF-1 on PRL and GH promoter activity in GH4C1 and GH3 cells. The cells were transiently transfected with 10 µg of the construct -3000Prl-CAT containing the 5' flanking region of the rat prolactin promoter (panel A), or with the same amount of the construct -530GH-CAT, which contains the rat GH promoter (panel B). CAT activity was determined in cells treated for 48 h with medium alone (-) or in the presence of 13 nM IGF-1 and/or 10 µM Fk. The data represent the mean ± SD obtained from three independent transfections.

 
According to the data presented in Figs. 1–3GoGoGo, where IGF-1 decreased PRL-mRNA levels in GH3 cells, it was expected that IGF-1 should decrease PRL promoter activity in these cells. However, we have not been able to find regulation of the PRL promoter by IGF-1 in transient transfection assays. As shown in panel A, Fk stimulated the PRL promoter, but IGF-1 was ineffective in regulating either basal activity or the response to the activator in GH3 cells.

The stimulatory effect of IGF-1 on the PRL promoter in GH4C1 cells was specific for this promoter. Figure 4BGo shows that the growth factor does not significantly induce GH promoter activity in GH4C1 cells. This promoter was strongly activated by T3, and IGF-1 did not alter this response. Additionally, there was not a significant inhibition of GH promoter activity by IGF-1 in this cell line (Fig. 4BGo), despite the finding that basal and induced levels of GH transcripts were significantly reduced.

It was conceivable that, if insulin receptors are expressed at substantial levels in GH4C1 cells, the stimulation of the PRL promoter by IGF-1 may reflect binding of IGF-1 to the insulin receptor. However, as shown in Fig. 5Go, this possibility can be dismissed. IGF-1 caused a dose-dependent stimulation of the -3000Prl-CAT construct in GH4C1 cells, with a half-maximal response found at approximately 2 nM. However, incubation with insulin, at concentrations similar to those of IGF-1, was unable to affect the PRL promoter in these cells. Neither IGF-1 nor insulin had a significant effect on the activity of the PRL promoter in GH3 cells (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Insulin does not regulate PRL promoter activity in GH4C1 cells. The cells were transfected with 10 µg of the construct -3000Prl-CAT and incubated with increasing concentrations of IGF-1 (•) or insulin ({circ}). CAT values were determined after 48 h of treatment in duplicate cultures and are expressed as fold-induction over the basal levels obtained in untreated cells.

 
One potential mechanism for the differences in the regulation of PRL promoter activity between both cell lines could be that GH3 cells may have inadequate levels of IGF-1 receptors, relative to GH4C1 cells. To test this possibility, both cell types were cotransfected with the -3000Prl-CAT construct and an expression vector for the IGF-1 receptor. Upon overexpression of the receptor, IGF-1 strongly increased promoter activity (approximately 16-fold) in GH4C1 cells, whereas the growth factor had little, if any, effect in GH3 cells (Fig. 6Go). These data show that high levels of IGF-1 receptors are not sufficient to activate the PRL promoter in GH3 cells and that the amount of IGF-1 receptors is limiting in GH4C1 cells.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. Influence of overexpression of the IGF-1 receptor on the activity of the PRL promoter. GH4C1 and GH3 cells were transfected with 10 µg of the PRL promoter construct in the presence or absence of 2 µg of an expression vector encoding the IGF-1 receptor (IGF-R). The cells were treated or not with 13 nM IGF-1 for 48 h, as indicated, and CAT activity determined. The data represent the mean ± SD from triplicate cultures.

 
IGF-1 does not alter the half-life of PRL or GH mRNAs in GH3 cells
Because IGF-1 strongly decreases GH and PRL mRNA without significantly affecting the activity of the constructs containing the 5'-flanking region of the PRL gene, it was possible that the main effect of IGF-1 was exerted at a posttranscriptional level by decreasing mRNA half-life. To test this possibility, GH and PRL-mRNA levels were determined in control GH3 cells and in cells incubated with IGF-1, T3, or the combination of both for 48 h and then incubated for varying times with 5 µg/ml actinomycin D, an inhibitor of RNA synthesis (Fig. 7Go). In a group of cells that did not receive the inhibitor, PRL and GH transcripts remain stable during 30 h, the longest time period studied (data not shown). Right panel shows that PRL-mRNA is very stable and disappeared with a similar half-life in both untreated and IGF-1-treated cells. The stimulatory effect of T3 did not reflect stabilization of PRL transcripts, which have a calculated half-life of approximately 17 h, both in the presence and absence of IGF-1 (right panel). GH-mRNA also was very stable in GH3 cells, with a similar half-life (22 h), regardless of the different treatments (left panel). Therefore, the decrease in GH and PRL transcripts in GH3 cells is not secondary to destabilization of these mRNAs on incubation with IGF-1.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 7. IGF-1 does not alter the half-life of GH or PRL transcripts in GH3 cells. The cells were first incubated with 5 nM T3 and/or 13 nM IGF-1 for 48 h. The culture medium was changed (time 0) and the cells incubated with medium containing 5 µg/ml actinomycin D. At the times indicated, RNA was extracted from duplicate cultures of control cells ({circ}) or cells treated with IGF-1 (•), T3 ({square}), or IGF-1 + T3 ({blacksquare}). Left panel, Levels of GH-mRNA; right panel, levels of PRL-mRNA. The data are expressed as percentages of the levels obtained at time 0 for each treatment.

 
Effect of activated- and dominant-negative Ras mutants on the PRL promoter
It has been demonstrated that oncogenic Ras significantly increases PRL promoter activity in pituitary cell lines, and we have evidence that endogenous Ras is required for induction of the activity of the PRL promoter by IGF-1 in GH4C1 cells. Because IGF-1 does not activate the PRL promoter in GH3 cells, there was the possibility that a component of the Ras signaling pathway was inactive in these cells. Figure 8Go compares the effect of oncogenic RasVal12 on PRL promoter activity in GH4C1 and GH3 cells treated with IGF-1 alone or in combination with Fk. Expression of oncogenic Ras strongly activated the PRL promoter in GH4C1 cells (panel A). Interestingly, IGF-1 further increased promoter activity in the presence of oncogenic Ras. Although IGF-1 was unable to regulate PRL promoter activity in GH3 cells, expression of oncogenic RasVal12 strongly induced the activity of the PRL CAT plasmid in these cells (panel B), showing that the signaling pathway downstream of Ras is intact in these cells. As expected (29), Fk did not stimulate the promoter in GH4C1 or GH3 cells expressing Ras, and in agreement with the data shown in Fig. 4AGo, the response to IGF-1 (expressed as fold-induction) decreased in Fk-treated cells. The dominant negative RasAsn17 mutant blocked specifically the induction caused by IGF-1 in GH4C1 cells, confirming that activation of the PRL promoter by this factor requires functional endogenous Ras; but this mutant did not affect the PRL promoter in GH3 cells, where IGF-1 was inactive.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 8. Ras activates the PRL promoter in GH3 and GH4C1 cells. The cells were cotransfected with 10 µg of 3000Prl-CAT alone or in combination with 10 µg of a vector expressing the oncogenic Ha-rasVal12, or with 30 µg of an expression vector for the dominant negative Ras mutant RasAsn17. CAT activity was determined 48 h after transfection in control cells (-) and in cells treated with 10 µM forskolin (Fk) in the presence (black bars) or absence (light bars) of 13 nM IGF-1. The data are the mean of the values obtained from two independent transfections with variation less than 10–15%.

 
Stimulation of MAP-K activity by IGF-1 in GH3 and GH4C1 cells
The lack of stimulation of the PRL gene by IGF-1 in GH3 cells could reflect uncoupling of the receptor from the Ras/MAPK pathway. If this were the case, a downstream component of this pathway, such as the MAP-K, should not be activated in response to IGF-1 in these cells. Alternatively, this response could be preserved in GH3 cells; and an additional pathway, different from the Ras/MAPK pathway, also required for PRL gene regulation by IGF-1, would be lost. We have therefore examined the MAP-K response to IGF-1 in GH3 cells. Figure 9Go compares MAP-K activity obtained in lysates from GH3 and GH4C1 cells incubated with IGF-1 for increasing time periods. IGF-1 produced a rapid and transient MAP-K activation in GH3 cells that was not significantly different from that found in GH4C1 cells. In both cell types, a maximal effect was found at 2–6 min, and the values returned to normal after 10 min. An incubation with 100 nM TPA for 10 min was used as a control of MAP-K activation in these cells. At this time, TPA caused a stimulation that was at least two-fold stronger that the maximal IGF-1 induction.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 9. IGF-1 activates MAP-K activity in GH3 and GH4C1 cells. GH4C1 and GH3 cells were treated with 13 nM IGF-1 for the times indicated or with 100 nM TPA for 10 min. Cell extracts were incubated with an anti-ERK-2 antibody, and kinase activity in the immunoprecipitates was determined with {gamma}-32P-ATP and MBP as the substrate. 32P incorporation into MBP was quantitated by densitometry after separation by electrophoresis in polyacrylamide gels.

 
Influence of IGF-1 on GHF-1/Pit-1 expression in GH4C1 and GH3 cells
The different behavior of the PRL and GH genes in GH4C1 and GH3 cells could be related to a different regulation of GHF-1/Pit-1 gene expression by IGF-1. For instance, the decrease in PRL and GH transcripts in IGF-1-treated GH3 cells could be secondary to a reduction in the concentration of GHF-1/Pit-1 that activates both genes. To test this hypothesis, Western blot analysis was performed with extracts from GH4C1 and GH3 cells. Proteins obtained from control cells and from cells treated with 13 nM IGF-1 for 48 h were assayed using a specific anti-GHF-1/Pit-1 antibody. Figure 10Go (panel A) shows that the concentration of the 33-kDa protein doublet of GHF-1/Pit-1 protein was decreased in GH3 cells treated with IGF-1. Quantification of this blot and additional experiments showed that treatment with IGF-1 reduced, by 30–50%, GHF-1/Pit-1 levels in GH3 cells. However, IGF-1 did not decrease the abundance of this transcription factor in GH4C1 cells.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 10. IGF-1 decreases GHF-1/Pit-1 expression in GH3 cells. Panel A, The levels of GHF-1/Pit-1 protein were determined by immunoprecipitation with an anti-GHF-1 antibody in untreated (-) GH4C1 and GH3 cells, and in cells treated with 13 nM IGF-1 for 48 h; panel B, GHF-1/Pit-1 mRNA levels were determined by Northern blot in GH4C1 and GH3 cells treated as in panel A. The sizes of the GHF-1/Pit-1 transcripts found are indicated with arrows.

 
The influence of IGF-1 on GHF-1/Pit-1 mRNA levels also was determined in both cell types. Figure 10Go (panel B) shows that two major classes of GHF-1/Pit-1 transcripts were found: a more prominent 3.1-kb band and a less abundant band of 1.2-kb. A fainter transcript of approximately 4 kb also was often detected. In agreement with the GHF-1/Pit-1 protein concentrations, the level of GHF-1/Pit-1 transcripts was reduced by IGF-1 in GH3 but not in GH4C1 cells. As in the case of the PRL and GH promoters, the activity of the GHF-1/Pit-1 promoter in transient transfection assays was not reduced by IGF-1 in GH3 cells (data not shown).

Figure 11Go shows the levels of GHF-1/Pit-1 transcripts in GH4C1 and GH3 cells treated with T3, RA, and Fk in the presence and absence of IGF-1. As illustrated in panel A, in GH3 cells, the inhibition of GHF-1/Pit-1 transcripts by IGF-1 was less marked than that caused by T3, and the combination of both did not produce further decreases. In addition, IGF-1-mediated reduction of GHF-1/Pit-1 mRNA levels was significantly attenuated in the presence of RA or Fk. Figure 11BGo shows that IGF-1 alters neither basal levels of GHF-1/Pit-1 mRNA nor RA-dependent stimulation or T3-dependent inhibition in GH4C1 cells.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 11. Regulation of GHF-1/Pit-1 mRNA levels by IGF-1, T3, RA, and forskolin in GH4C1 and GH3 cells. RNA obtained from GH3 cells (panel A) and GH4C1 cells (panel B) was used for Northern blot analysis with the GHF-1/Pit-1 cDNA probe. The RNA was obtained from control untreated cells and from cells incubated with 5 nM T3, 1 µM RA, or 10 µM forskolin (Fk) in the presence or absence of 13 nM IGF-1, as indicated.

 
The lack of response of the PRL promoter to IGF-1 in GH3 cells may reflect the presence of limiting amounts of GHF-1/Pit-1 in these cells. To directly test this hypothesis, the effect of IGF-1 on the -3000Prl-CAT construct also was examined in GH3 and GH4C1 cells transfected with an expression vector encoding GHF-1/Pit-1. As shown in Fig. 12Go, expression of GHF-1/Pit-1 had little, if any, effect on PRL promoter activity in GH4C1 cells. However, basal promoter activity increased in GH3 cells transfected with GHF-1/Pit-1; and under these conditions, IGF-1 did not decrease the activity of the PRL promoter but, instead, increased promoter activity by approximately 2-fold.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 12. Effect of expression of GHF-1 on the PRL promoter response to IGF-1. GH4C1 and GH3 cells were transfected with 10 µg of 3000Prl-CAT alone (-) or in combination with 10 µg of a vector expressing GHF-1. CAT activity was determined 48 h after transfection in control cells and in cells treated with 13 nM IGF-1. The data represent the mean ± SD of the values obtained from three independent cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work presents evidence of alternate inhibition or stimulation of the PRL gene by IGF-1 in two different rat pituitary cell lines. Whereas in GH4C1 cells, expression of this gene is strongly induced by IGF-1, in GH3 cells, the growth factor reduces PRL transcripts and represses the response to different PRL inducers, such as T3, RA, or Fk (28, 29, 30, 31). The GH gene, another pituitary-specific gene, also is differentially regulated by IGF-1. The expression of GH is not significantly affected by IGF-1 in GH4C1 cells but is reduced in GH3 cells. We have previously shown that RA induces GH gene transcription in GH1 and GH4C1 cells, and we have mapped the RA response element to the same sequences that mediate regulation by T3 (26, 27). Our present results show that RA and T3 also induce GH gene expression in GH3 cells and that this response is significantly attenuated by IGF-1. The reduction of GH and PRL mRNA levels, caused by IGF-1 in GH3 cells, could reflect a decrease of transcription or could be secondary to a posttranscriptional mechanism, leading to mRNA destabilization. The latter mechanism was eliminated, because the apparent half-life of GH and PRL mRNAs was not reduced in IGF-1-treated GH3 cells.

The expression of the GH and PRL genes is dependent on the binding of GHF-1/Pit-1 to their promoters. We have found that IGF-1 decreases the amount of GHF-1/Pit-1 in GH3 cells and that this decrease is secondary to a reduction in GHF-1/Pit-1 mRNA levels. This observation is in agreement with a recent report (32) showing that IGF-1 inhibited basal GHF-1/Pit-1 mRNA concentration and blunted the response to GH-RH in primary cultures of rat anterior pituitary cells. However, GHF-1/Pit-1 levels were not reduced by IGF-1 in GH4C1 cells. Furthermore, we have previously described that whereas T3 decreases (33), RA increases (34) GHF-1/Pit-1 mRNA levels, and these responses are not modified by IGF-1 in GH4C1 cells.

The reduction in the concentration of the transcription factor that is required for the expression of both the GH and PRL genes is certainly in agreement with the decreased levels of their transcripts observed in IGF-1-treated GH3 cells. Unexpectedly, the activity of the PRL and GH promoters, which contain the binding sites for GHF-1/Pit-1, was not inhibited by IGF-1 in GH3 cells in transient transfection assays. Although we do not dismiss the possibility that the sequences responsible for the inhibitory effect of IGF-1 on pituitary gene expression in GH3 cells could be outside the promoter fragments examined, it seems likely that the results obtained in the transient transfection assays do not exactly reflect the behavior of the endogenous gene. Discrepancies between the regulation of the endogenous PRL gene and the transiently transfected promoter have been previously reported to occur for the regulation of this gene by insulin. It has been found that insulin (as observed by us with IGF-1 in these cells) stimulates expression of the endogenous gene but, under identical conditions, does not stimulate PRL-CAT plasmids (35, 36). The authors attribute the lack of response of the transfected gene to the existence of low levels of insulin receptors in these cells, because cotransfection with an insulin receptor expression plasmid was required to observe insulin stimulation of the PRL promoter. Furthermore, it has been previously shown (37) that GH3 cells are relatively resistant, as compared with GC cells, to the action of IGF-1 because they contain a lower number of IGF-1 receptors. Therefore, it was likely that the concentration of the IGF-1 receptors may be insufficient in GH3 cells to cause a reduction in the activity of the GH and PRL promoters in transient transfection assays. However, this possibility can be dismissed, because the overexpression of IGF-1 receptors does not confer a significant promoter response to IGF-1 in GH3 cells. In contrast, IGF-1 stimulation of the PRL promoter is observed in GH3 cells transfected with an expression vector encoding GHF-1/Pit-1. This result, which further suggests a role of the endogenous GHF-1/Pit-1 concentration in the differential response to IGF-1, is in agreement with the finding that overexpression of the transcription factor also enhances activation of the PRL promoter by Ras in GH4C1 cells (12).

In contrast to the findings in GH3 cells, the PRL promoter is significantly activated by IGF-1 in GH4C1 cells, and this response is further enhanced upon overexpression of the IGF-1 receptor. Stimulation by the growth factor in GH4C1 cells requires activation of the Ras/MAP-K pathway, because it is blocked by a dominant negative Ras mutant. It was then possible that some component of this pathway was defective in GH3 cells. Our results show that the pathway downstream of Ras is intact, because oncogenic Ras clearly activates the PRL promoter in GH3 cells. It also was possible that a component linking the IGF-1 receptor to Ras was uncoupled in these cells. If this were the case, IGF-1 should not activate this signaling pathway. However, the growth factor transiently stimulated MAP-K activity in GH3 cells. Both the intensity and the time-course of the activation were similar in GH3 and GH4C1 cells. From these results, it can be deduced that the defect occurs in other pathway(s), in addition to the Ras/MAP-K cascade. Although IGF-1 is a potent growth factor for other cell types, it exerts little mitogenic effects in pituitary cells (6). The rapid induction of MAPK activity, after incubation with growth factors, is followed by a slower and persistent late phase of activity that correlates with mitogenic activity, and no second phase of activation is observed in GC, GH4C1, or GH3 cells (Ref. 6, and this study). Therefore, the influence of IGF-1 on pituitary gene expression should not merely reflect changes in cell growth and the cell cycle.

The existence of an additional signaling pathway that diverges after receptor activation and contributes to the stimulation of the PRL promoter by IGF-1 is suggested by the finding that, in GH4C1 cells, IGF-1 can further increase the activity of the PRL promoter in the presence of oncogenic Ras. It is interesting that EGF, also a ligand of a tyrosine kinase receptor, activates the PRL promoter by a Ras-independent mechanism in pituitary GH4 cells (38). The exact mechanism by which this factor regulates the PRL gene is still unknown, but it is well established that ligand-activation of different receptor tyrosine kinases allows several proteins to become associated with specific phosphotyrosines and to initiate different signaling cascades. Our results are compatible with a model in which IGF-1 uses both the Ras/MAP-K and other pathway(s) to activate the IGF-1 gene. Stimulation of such pathways would be required for the IGF-1 action. GH3 cells would be deficient in the second pathway and, therefore, in stimulation of the PRL promoter, whereas GH4C1 cells would respond to IGF-1 by stimulating both pathways and, consequently, the PRL gene. That the second pathway is alone insufficient for this stimulation is shown by the finding that the dominant negative Ras mutant abolishes the IGF-1 response in GH4C1 cells. The finding that oncogenic Ras, but not IGF-1, is able to activate the PRL promoter in GH3 cells suggests that a strong and sustained stimulation of Ras, that is not obtained when the endogenous Ras is transiently activated by IGF-1, is required to achieve PRL promoter activation.

Although different pituitary cell lines are normally indistinctly used in studies of hormonal regulation of gene expression, there are previous reports of differential regulation in different pituitary cell lines. This is especially true for the PRL gene. For instance, T3 decreases PRL gene expression in GH1 cells, whereas it increases PRL transcripts in GH3 and GH4C1 cells (Refs. 21 and 30, and this study). Furthermore, insulin has been shown to stimulate the PRL gene in GH4C1 cells (31, 35, 39) without affecting PRL expression in primary pituitary cultures (8). Pituitary gene response to IGF-1 in primary cultures of rat anterior pituitary cells is rather similar to that found by us in GH3 cells and strikingly different to that observed in GH4C1 cells. IGF-1 has an inhibitory effect on GH and GHF-1/Pit-1 gene expression in GH3 and normal pituitary cells, but it has a specific stimulatory effect on PRL transcription without affecting GH or GHF-1/Pit-1 expression in GH4C1 cells. The different behavior of the two cell lines could be related to the fact that, although both are tumor cells, the GH3 cells are not variant cell lines and thus are presumably one step closer than GH4C1 cells to their normal counterparts. Regulation of pituitary gene expression provides a powerful tool for future studies on the delineation of the molecular mechanisms by which the same hormone or growth factor can repress or stimulate the same gene in closely related cell types.

Finally, the action of IGF-1 in the pituitary axis must be complex and should take into account other factors such as IGF-2 or the IGF-binding proteins (IGFBPs). Although our experiments have been performed in the absence of serum, how each of the different binding proteins function to augment or inhibit the effect of IGF-1 in pituitary gene expression, or how IGF-1 works in conjunction with IGF-2 or other factors, has not yet been delineated and will require future studies.


   Footnotes
 
1 This work was supported by the Comunidad de Madrid and by Grant PB-94-0094 from the Dirección General de Investigación Cientifica y Técnica. Back

Received February 13, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Karin M, Castrillo JL, Theill LE 1990 Growth hormone gene regulation: a paradigm for cell-type-specific gene activation. Trends Genet 6:92–96[CrossRef][Medline]
  2. Voss JW, Rosenfeld MG 1992 Anterior pituitary development: short tales from dwarf mice. Cell 70:527–530[CrossRef][Medline]
  3. Rosenfeld RG, Ceda G, Cutler CW, Dollar RA, Hoffman AR 1986 Insulin and insulin-like growth factor (somatomedin) receptors on cloned rat pituitary tumor cells. Endocrinology 117:2008–2116[Abstract]
  4. Jones JI, Clemmons DR 1995 Insulin-like growth factor and their binding proteins: biological actions. Endocr Rev 16:3–34[CrossRef][Medline]
  5. LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin-like growth factor-1 receptor. Endocr Rev 16:143–163[CrossRef][Medline]
  6. Webster J, Prager D, Melmed S 1994 Insulin-like growth factor-1 activation of extracellular signal-related kinase-1 and -2 in growth hormone-secreting cells. Mol Endocrinol 8:539–544[Abstract]
  7. Yamashita S, Melmed S 1986 Insulin-like growth factor-1 action on hypothyroid rat pituitary cells: suppression of triiodothyronine-induced growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 118:1483–1490[Abstract]
  8. Yamashita S, Melmed S 1987 Insulinlike growth factor 1 regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 79:449–452
  9. Prager D, Melmed S 1988 Insulin regulates expression of the human growth hormone gene in transfected cells. J Biol Chem 263:16589–16585
  10. Deleted in proof
  11. Conrad KE, Oberweter JM, Vaillancourt R, Johnson GL, Gutierrez-Hartmann A 1994 Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression. Mol Cell Biol 14:1553–1565[Abstract/Free Full Text]
  12. Bradford AP, Conrad KE, Wasylyk C, Wasylik D, Gutierrez-Hartmann A 1995 Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediated Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain. Mol Cell Biol 15:2849–2857[Abstract]
  13. Howard PW, Maurer RA 1995 A composite Ets/Pit-1 binding site in the prolactin gene can mediate transcriptional responses to multiple signal transduction pathways. J Biol Chem 270:20930–20936[Abstract/Free Full Text]
  14. Bancroft C, Gick GG, Johnson ME, White BA 1985 Regulation of growth hormone and prolactin gene expression by hormones and calcium. Biochem Actions Horm 12:173–213
  15. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  16. Gubbins EJ, Maurer RA, Lagrimini M, Erwin CR, Donelson JE 1980 Structure of the rat prolactin gene. J Biol Chem 255:8655–8662[Abstract/Free Full Text]
  17. Seeburg PH, Shine J, Martial J, Baxter JD, Goodman HM 1977 Nucleotide sequence and amplification in bacteria of the structural gene for rat growth hormone. Nature 27:486–489
  18. Bodner N, Karin M 1987 A pituitary-specific transacting factor can stimulate transcription from the growth hormone promoter in extracts of non expressing cells. Cell 50:267–275[CrossRef][Medline]
  19. Flug F, Copp RP, Casanova J, Horowitz DZ, Janocko L, Plotnick M, Samuels HH 1987 Cis-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone. J Biol Chem 262:6373–6382[Abstract/Free Full Text]
  20. McCormick A, Brady H, Theill LE, Karin M 1990 Regulation of the pituitary-specific homeobox gene GHF-1 by cell autonomous and environmental cues. Nature 345:829–832[CrossRef][Medline]
  21. Stanley F 1989 Transcriptional regulation of prolactin gene expression by thyroid hormone-alternate suppression and stimulation in different GH cell lines. Mol Endocrinol 3:1627–1633[CrossRef][Medline]
  22. Cales C, Hancock JF, Marshall CJ, Hall A 1988 The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature 332:548–551[CrossRef][Medline]
  23. Feig L, Cooper GM 1988 Inhibition of NIH 3T3 cell proliferation by a mutant Ras protein with preferential affinity for GDP. Mol Cell Biol 8:3235–3243[Abstract/Free Full Text]
  24. Theill LE, Hattori K, Domenico D, Castrillo JL, Karin M 1992 Differential splicing of the GHF1 primary transcript gives rise to two functionally distinct homeodomain proteins. EMBO J 11:2261–2269[Medline]
  25. Kato H, Faria TS, Stannard B, Roberts CT, LeRoith D 1993 Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-1 (IGF-1) receptor. J Biol Chem 268:2655–2661[Abstract/Free Full Text]
  26. Bedo G, Santisteban P, Aranda A 1989 Retinoic acid regulates growth hormone gene expression. Nature 339:231–234[CrossRef][Medline]
  27. Garcia-Villalba P, Au-Fleigner M, Samuels HH, Aranda A 1993 Interaction of thyroid hormone and retinoic acid receptors on the regulation of the rat growth hormone gene promoter. Biochem Biophys Res Commun 191:580–586[CrossRef][Medline]
  28. Maurer RA 1981 Transcriptional regulation of the prolactin gene by ergocryptine and cyclic AMP. Nature 294:94–97[CrossRef][Medline]
  29. Conrad KE, Gutierrez-Hartmann A 1992 The ras and protein kinase A pathways are mutually antagonistic in regulating rat promoter prolactin activity. Oncogene 7:1279–1286[Medline]
  30. Day RN, Maurer RA 1989 Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation. Mol Endocrinol 3:931–938[CrossRef][Medline]
  31. Stanley FM 1992 An element in the rat prolactin promoter mediates the stimulatory effect of insulin on transcription of the prolactin gene. J Biol Chem 267:16719–16726[Abstract/Free Full Text]
  32. Soto JL, Castrillo JL, Dominguez F, Dieguez C 1995 Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture. Endocrinology 136:3863–3870[Abstract]
  33. Sanchez-Pacheco A, Palomino T, Aranda A 1995 Negative regulation of expression of the pituitary-specific transcription factor GHF-1/Pit-1 by thyroid hormones through interference with promoter enhancer elements. Mol Cell Biol 15:6322–6330[Abstract]
  34. Sanchez-Pacheco A, Palomino T, Aranda A 1995 Retinoic acid induces expression of the transcription factor GHF-1/Pit-1 in pituitary prolactin- and growth hormone-producing cells. Endocrinology 136:5391–5398[Abstract]
  35. Jacob KK, Stanley FM 1994 The insulin and cAMP response elements of the prolactin gene are overlapping sequences. J Biol Chem 269:25515–25520[Abstract/Free Full Text]
  36. Pickett CA, Gutierrez-Hartmann A 1994 Ras mediates Src but not epidermal growth factor-receptor tyrosine kinase signaling pathways in GH4 neuroendocrine cells. Proc Natl Acad Sci USA 91:8612–8616[Abstract/Free Full Text]
  37. Yamasaki H, Prager D, Gebremedhin S, Moise L, Melmed S 1991 Binding and action of insulin-like growth factor 1 in pituitary tumor cells. Endocrinology 128:857–862[Abstract]
  38. Pickett CA, Gutierrez-Hartmann A 1995 Epidermal growth factor and ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways. Mol Cell Biol 15:6777–6784[Abstract]
  39. Stanley FM 1988 Stimulation of prolactin gene expression by insulin. J Biol Chem 263:13444–13448[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J Mol EndocrinolHome page
R. M Luque, M. D Gahete, R. J Valentine, and R. D Kineman
Examination of the direct effects of metabolic factors on somatotrope function in a non-human primate model, Papio anubis.
J. Mol. Endocrinol., August 1, 2006; 37(1): 25 - 38.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
F.-y. Gong, Y.-f. Shi, and J.-y. Deng
The regulatory mechanism by which interleukin-6 stimulates GH-gene expression in rat GH3 cells.
J. Endocrinol., August 1, 2006; 190(2): 397 - 406.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
M Fernandez, F Sanchez-Franco, N Palacios, I Sanchez, and L Cacicedo
IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1
J. Mol. Endocrinol., June 1, 2005; 34(3): 699 - 712.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
R. Salvatori, X. Fan, P. E. Mullis, A. Haile, and M. A. Levine
Decreased Expression of the GHRH Receptor Gene Due to a Mutation in a Pit-1 Binding Site
Mol. Endocrinol., March 1, 2002; 16(3): 450 - 458.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Fruchtman, L. Jackson, and R. Borski
Insulin-Like Growth Factor I Disparately Regulates Prolactin and Growth Hormone Synthesis and Secretion: Studies Using the Teleost Pituitary Model
Endocrinology, August 1, 2000; 141(8): 2886 - 2894.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. P. Bradford, K. S. Brodsky, S. E. Diamond, L. C. Kuhn, Y. Liu, and A. Gutierrez-Hartmann
The Pit-1 Homeodomain and beta -Domain Interact with Ets-1 and Modulate Synergistic Activation of the Rat Prolactin Promoter
J. Biol. Chem., February 4, 2000; 275(5): 3100 - 3106.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. Stefaneanu, L. Powell-Braxton, W. Won, V. Chandrashekar, and A. Bartke
Somatotroph and Lactotroph Changes in the Adenohypophyses of Mice with Disrupted Insulin-Like Growth Factor I Gene
Endocrinology, September 1, 1999; 140(9): 3881 - 3889.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Castillo, A. I.
Right arrow Articles by Aranda, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Castillo, A. I.
Right arrow Articles by Aranda, A.

HOME HELP FEEDBACK