1 Department of Medicine, Mount Sinai Hospital and University of Toronto, 2 Department of Pathology, University Health Network and University of Toronto, The Freeman Centre for Endocrine Oncology, and The Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9
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ABSTRACT |
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Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of activities. Signaling of the 23 members of the FGF family is mediated through FGFR1-4. We show that FGF-19, which selectively binds FGFR4, can induce prolactin (PRL) but not growth hormone expression. FGF-19 also stimulated MAPK activation, an effect that was abrogated by a soluble dominant negative (dn) form of FGFR4. The response of the pituitary PRL promoter to FGF maps to an Ets-Pit1 binding site. We have previously shown that the hematopoietic zinc finger-containing transcription factor Ikaros (Ik) regulates FGFR4 as part of an overlapping site with that for an Ets-type factor in the FGFR4 promoter. Thus, we examined whether FGF-19 might regulate its own receptor through the Ets-Ik element in the FGFR4 promoter. Ets stimulated and dn-Ets inhibited basal FGFR4 and PRL promoter activity. In contrast, Ets enhanced FGF-19-induced PRL activation but failed to confer an effect for FGF-19 on the FGFR4 promoter. We conclude that FGFR4 mediates FGF-19 signaling to the PRL promoter. Our data also suggest a possible functional role for Ik in sorting Ets signals to the FGFR4 promoter, as distinct from the PRL promoter, where Ets partners with Pit1.
fibroblast growth factor receptor 4; fibroblast growth factor-19; pituitary; Ikaros; Ets1
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INTRODUCTION |
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THE PITUITARY IS THE SITE of synthesis and the target of fibroblast growth factors (FGFs) that modulate hormone production and cell proliferation (6). FGF-2 (bFGF) was originally identified in bovine pituitary and is overexpressed in pituitary tumors, with the highest levels noted in aggressive tumors (7, 8). FGF-4 has also been found in transforming DNA sequences from human prolactin (PRL)-secreting pituitary tumors (12). FGF ligands have also been shown to stimulate PRL secretion (6). Little is known, however, about the mechanism of this effect and the receptors involved in mediating it.
FGF signaling is potentially mediated through one of four FGFRs, a complex family of transmembrane receptor tyrosine kinases (11). Each prototypic receptor is composed of three immunoglobulin (Ig)-like extracellular domains, two of which are involved in ligand binding, a single transmembrane domain, a split tyrosine kinase, and a COOH-terminal tail with multiple autophosphorylation sites (11). We have recently shown that targeted expression of a truncated pituitary tumor-derived FGFR4 (ptd-FGFR4) to the pituitary results in tumor formation (10). In marked contrast to the oncogenic effects of ptd-FGFR4, expression of full-length wild-type FGFR4 failed to stimulate adenoma formation in the pituitaries of transgenic mice (10). In the current study, we sought to examine other functions of full-length FGFR4 in the pituitary.
Although multiple FGFs interact with each of the four FGFRs, we took advantage of FGF-19 as a novel high-affinity heparin-dependent ligand that shows exclusive binding to FGFR4 (23) to analyze FGFR4 function in a complex cell system. In addition to demonstrating an effect on MAPK activation and PRL gene expression, we investigated the molecular mechanisms of FGFR4 signaling to the PRL promoter and compared them with those to the FGFR4 promoter itself. We present evidence that a member of the Ets family of transcription factors is a functional nuclear target that sorts and integrates distinct FGFR4-generated MAPK signals to the PRL and FGFR4 promoters.
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MATERIALS AND METHODS |
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Cell culture. The rat pituitary GH4C1 cell and the human embryonic kidney HEK 293 cell line were propagated in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) with high glucose supplemented with 10% fetal bovine serum (FBS; Sigma, Oakville, ON), 2 mM glutamine, 100 IU/ml of penicillin, and 100 µg/ml of streptomycin. Twenty-four hours before transfection, cells were plated with DMEM containing 10% serum. Primary human pituitary samples were obtained at the time of transsphenoidal pituitary surgery, as previously described (3).
Plasmids.
Promoter analysis of the human FGFR4 gene was performed as previously
described (24). Full-length human FGFR4 (10)
and soluble dominant negative FGFR4 (sFGFR4) (9) were used
as previously described. The orientation and sequence of all constructs
were verified by restriction analysis and nucleotide sequencing. The expression vectors pSG5-Ets-1, encoding the chicken Ets1 isoform, and
pAPr EtsZ, encoding dominant negative Ets (dn-Ets), were obtained from
Drs. A. Bradford (University of Colorado) and M. Ostrowski (Ohio State
University) (20). PRL and growth hormone (GH) promoter activity was analyzed with reporter constructs pSV2A-rPRL-Luc containing the 422-bp fragment of the rat rPRL promoter or
320-bp of the rGH promoter, respectively (kindly provided by Dr. H. Elsholtz, University of Toronto). To normalize for transfection efficiency variation within and between experiments, 20 ng/well of
pSV-
-galactoside control vector (Promega, Madison, WI) were included
with each transfection. The results were normalized to
-galactosidase activity.
Transfection and luciferase assays.
All plasmid reporters were prepared by column chromatography (QiaGen,
Missisauga, ON) for sequencing and transfections. Cells were
transfected by the lipofectamine method (GIBCO) according to the
manufacturer's protocol. Cells were plated into six-well cluster
dishes (7 × 105 cells per well), transfected on the
following day with 3 µl or 5 µl/well of lipofectamine and 1 or 2 µg of DNA per well, as indicated. The total amount of transfected DNA
was kept constant by adding an empty vector. Transfection efficiency
was monitored by simultaneous co-transfection with a -galactosidase
control expression plasmid CMV-
gal (20 ng/well). Forty-eight hours
after transfection, cells were lysed in buffer containing 25 mM
glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1% Triton X, and 1 mM DTT. Luciferase activity was measured for 20 s in a
luminometer.
-Galactosidase activity was measured to normalize for
variations in transfection efficiency. Promoter activity of each
construct was expressed as firefly luciferase per
-galactosidase
activity. Each experiment was independently performed on three separate
occasions with triplicate wells in each experiment.
Stimulation and hormone regulation.
Stimulation of PRL by FGFs was analyzed in GH4 cells grown in
6-multiwell microtiter plates (5 × 104 cells/well),
preincubated for 48 h in serum-free defined media (insulin 5 µg/ml, transferrin 5 µg/ml). Treatments were based on time- (48
h) and dose (
100 ng/ml)-response studies. Time and dose peak effects
were utilized for further response studies with FGF-1 (Sigma, 50 ng/ml) or FGF-19 (Dr. A. L. Gurney, Genentech, San Francisco, CA;
50 ng/ml) and 10 U/ml of heparin carried out in serum-free medium for
24 h at 37°C.
mRNA analysis. Total RNA was extracted by the guanidinium isothiocyanate method. One microgram of DNase-treated RNA was used for reverse transcription. This was performed with 2.5 U/ml of murine leukemia virus reverse transcriptase, 2.5 mM MgCl2, 1 mM dNTP, 2.5 mM random hexamers, and 1 U/ml of RNase inhibitor. The integrity of RNA from each sample was assessed by amplification of the PGK-1 housekeeping gene, as previously described (15). PCR analyses were performed with the following primers: forward 5'-GGTGGTCCACGTATGGATCCTG-3', corresponding to nucleotides 481-503, and reverse 5'-GGGCGAAGAGAACATGTCAG-3', corresponding to nucleotides 1036-1017 of the human FGF-19 cDNA sequence (GenBank accession no. AF110400) to amplify a 556-bp product. Primers were designed to span at least one intron to permit the exclusion of genomic DNA contamination. PCR conditions were optimized to ensure product linearity. The identity of products was confirmed by sequencing.
For Northern blotting, 20 µg of DNase-treated RNA from treated and control cells were electrophoresed on 0.9% agarose gels, transferred to a nylon membrane, fixed with a UV crosslinker, prehybridized, and hybridized at 55°C with the rPRL or GAPDH cDNA. Blots were washed and autoradiographed for 24-48 h.Western blot analysis. Protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein (50 µg) from cell lysates or media were solubilized in 2.5× SDS sample buffer and separated on SDS 8% polyacrylamide gel and transferred to nitrocellulose. Apparent molecular weights were determined by comparison with concurrently electrophoresed standards. PRL and GH protein levels were determined using the following antibodies: polyclonal antisera to rPRL or GH [donated by the National Hormone and Pituitary Program (NHPP), National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Child Health and Human Development, Bethesda, MD] applied at dilutions of 1:8,000 and 1:50,000, respectively, or a polyclonal affinity-purified rabbit antiserum directed against the carboxy terminus of hFGFR4 (Santa Cruz). Immunoblotting with a monoclonal anti-His antibody (InVitrogen) was used to confirm His-tagged sFGFR4 expression. Total and phosphorylated MAPK was detected by specific antisera that recognize the p42/44 corresponding to Erk1/2 members of the MAPK family (New England Biolabs). An actin control was performed using a monoclonal antibody (Sigma) at 1:500. Blots were scanned for densitometric analysis.
Statistical analyses. Data are expressed as means ± SE. Differences were examined by one-way ANOVA or Student's t-test, both with a significance level of <0.05.
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RESULTS |
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Expression of FGF-19.
Although FGFR4 is expressed by pituitary GH4C1 cells (24),
we were not able to detect significant amounts of expression of its
selective ligand (23) FGF-19 in the pituitary. Figure 1 depicts RT-PCR analysis of human
pituitary specimens of different adenoma types. Despite a positive
signal for the housekeeping PGK-1 gene, these samples failed to yield a
product with FGF-19 primers. As previously described (23),
the human colon adenocarcinoma cell line SW480 displayed markedly
elevated levels of FGF-19 message (Fig. 1).
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FGF-19-mediated FGFR4 activation induces PRL expression.
We took advantage of the FGFR4-selective ligand FGF-19 to determine
whether it can activate FGFR4 and stimulate pituitary PRL expression.
Because the rodent FGF-19 has not yet been cloned, we used recombinant
human FGF-19 and transfected rat pituitary GH4 cells with the human
FGFR4 to establish the response to this FGF. Figure
2A depicts Western blotting
analysis of lysates from GH4 cells exposed to the non-FGFR-selective
FGF-1 (18) or FGF-19. Both compounds resulted in
significant PRL protein stimulation (Fig. 2A), with
densitometric analysis shown in Fig. 2B. In addition, untransfected GH4 cells also responded to FGF-19 treatment with PRL
protein (Fig. 2A) and to a lesser extent mRNA (Fig.
2C) induction, consistent with the known expression of FGFR4
by GH4 cells (24), providing evidence that human FGF-19
can activate the rodent FGFR4. In contrast to the effects of FGF-19 on
PRL, however, GH and FGFR4 protein expression was unaffected by either
FGF treatment (data not shown).
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FGFR4 transduces signaling to the PRL but not to the FGFR4
promoter.
Rat pituitary GH4 cells express PRL, whose basal and FGF-mediated
stimulation occurs through a well described bipartite complex formation
between Ets and Pit-1 (20). Figure
4A demonstrates that FGF-19
can activate the PRL promoter but not influence its own receptor FGFR4.
Treatment of GH4 cells with use of the non-FGFR4-selective ligand FGF-1
resulted in induction of PRL promoter activity by nearly eightfold. As
noted at the level of mRNA, the highly FGFR4-selective ligand FGF-19
resulted in a more modest twofold induction of PRL activity compared
with FGF-1. We suspect that this difference is determined, at least
partially, by the expression of multiple FGFRs in the pituitary
(1). Neither FGF-19 nor FGF-1 had influence on FGFR4
promoter activity, however (Fig. 4A; right).
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Ets binding sites contribute to PRL and FGFR4 basal promoter activity. We have previously shown that overexpression of Ets1 results in activation of the FGFR4 promoter, an effect that was attenuated with disruption of the overlapping Ik binding site (24). Co-transfection of Ets1 and Ik1 resulted in a greater degree of wild-type promoter stimulation compared with Ets1 alone, suggesting a functional synergism between these two factors in pituitary FGFR4 regulation (24). To further determine the significance of the Ets binding site within the FGFR4 minimal promoter, we compared the effect of co-transfection of wild-type Ets1 or a dn form of Ets on the FGFR4 as well as the PRL promoter. Figure 4B reveals that Ets1 transfection can activate either promoter by ~300%, whereas dn-Ets inhibits both promoters compared with vector control-transfected GH4 cells.
FGF-19 and FGF-1 enhance Ets1 signaling to the PRL but not to the FGFR4 promoter. Given the importance of MAPK in FGFR4 signaling, together with our findings on the role of Ets in regulating FGFR4 expression, we asked whether the Ik/Ets element may mediate FGF-19 responsiveness to the FGFR4 promoter. To answer this question, we examined the response of the FGFR4 and PRL promoters co-transfected with Ets1 and treated with FGF-1 or FGF-19 (Fig. 4C). Treatment with FGF-19 as well as FGF-1 resulted in further enhancement (2- and 6-fold, respectively) of Ets1-mediated activation of the PRL promoter (Fig. 4C, left). Moreover, dnEts resulted in attenuation of the FGF-19 and FGF-1 responses on the PRL promoter (Fig. 4C). Despite the effects of FGF-19 and FGF-1 on Ets1 activation of the PRL promoter, both FGF ligands failed to stimulate Ets1-induced FGFR4 activity (Fig. 4C, right). Similarly, co-transfection of Ik1 did not confer an effect for FGF-1 or FGF-19 on the FGFR4 promoter (data not shown). Moreover, no significant effects on GH gene activity were noted in the presence of FGF-1 or FGF-19 treatment, co-transfection of Ets1, or both FGF treatment and Ets1 co-transfection (data not shown).
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DISCUSSION |
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Our data identify a role for FGF-19 and its receptor FGFR4 in the pituitary. FGF-19 stimulates MAPK, which in turn phosphorylates and stimulates Ets. Ets binds the promoter in the PRL and the FGFR4 genes, but Ets has a pivotal role in sorting MAPK-generated signals through interaction with Pit-1 to the PRL promoter and through Ik to the FGFR4 promoter. These events confer selectivity of the unique responses generated by individual FGF-FGFR interactions.
The biological importance of the FGF family is underscored by the expanding number of ligands that now includes at least 23 members with varying mitogenic, angiogenic, and hormone-regulatory functions (4, 16). The FGFR domains involved in ligand binding are complex. FGFR1, -2, and -3 bind to or mediate responsiveness to both FGF-1 and FGF-2, whereas FGFR4 binds FGF-1 with 10-fold greater affinity than FGF-2 (19, 22). Although the prototypic receptor has three Ig-like domains, FGFR isoforms that lack the first Ig-like domain are still efficiently activated by FGFs (5); it has been suggested that the first domain may even decrease the binding affinity of FGFs and heparin. The second and third Ig-like domains are implicated in FGF binding in a highly specific fashion for each ligand: FGF-1 binds to loop 2 of FGFR2 but not to loop 3; FGF-7 binds to loop 3 but not to loop 2. FGF-7 binds to the loop 3 IIIb variant, whereas FGF-2 binds the IIIc variant. FGF-1 binds both variants equally (18). FGF-19, however, uniquely binds intact FGFR4 (23). We took advantage of this property to specifically define the role of FGFR4 in the pituitary that commonly expresses multiple types of FGFRs.
As with other tyrosine kinase receptors, FGFRs are activated by
dimerization, resulting in autophosphorylation and subsequent recruitment of intracellular signaling proteins, notably
phospholipase C- (PLC-
) (11, 17). A 28-amino acid
peptide containing Tyr766 of FGFR1 has been identified as
the major binding site for PLC-
; the analogous residue is conserved
in all FGFRs, with Tyr754 being the putative site in FGFR4.
Activated FGFR1 and FGFR4 homodimers interact with PLC-
(21). DNA synthesis and cell proliferation are equally
induced by FGFR1 and FGFR4 (21). Mutations in the activation loop of kinase domains of FGFR1, FGFR3, and FGFR4 have revealed that all three receptor domains can equally transform NIH 3T3
cells, induce neurite outgrowth in PC12 cells, and phosphorylate Shp2,
PLC-
, and MAPK (13). Using FGF-19 as a selective ligand for FGFR4, we now show that FGF-19 through intact FGFR4 can activate MAPK and that this response is associated with PRL induction.
FGFs have been shown to induce PRL gene transcription (20); however, the role of FGFR4 in mediating this response has not been identified, and, indeed, little is known about which FGF/FGFR interactions are involved in mediating FGF signaling in the pituitary. Previous studies using FGF-2 and FGF-4 in GH4 cells revealed that activation of the PRL promoter is independent of Ras and Raf-1 but requires MAPK (20). We thus sought to determine whether FGFR4 is involved in transducing the FGF signal to the PRL gene. We show, for the first time, that the FGFR4-selective ligand FGF-19 (23) can activate MAPK and the PRL promoter, thus defining a contribution for FGFR4 in mediating pituitary FGF signaling to the PRL promoter. As anticipated, PRL stimulation elicited by FGF-19 was not as robust as that induced by the non-FGFR-selective FGF-1 ligand, consistent with FGF-1-mediated effects through multiple FGFRs that are expressed in the pituitary (1). The only other previously known function of FGF-19 has been a synergistic interaction with Wnt-8c in initiating inner ear development (14). In contrast to the effects of FGF-19 on the PRL promoter, FGF-19 did not influence the regulation of its own receptor. These findings are in contrast to those in the FGFR2 promoter, where FGFR2 is downregulated in response to FGF ligand stimulation (2).
In our analysis of the FGFR4 promoter, we characterized a 214-bp
fragment of the 5' region that is important for activity. Further
mapping identified an ~40-bp fragment (64/
25) that demonstrated strong binding with nuclear extracts from pituitary GH4 cells (24). This fragment contained functional binding sites for
the zinc finger-containing transcription factor Ik flanked by two sites
for Sp1 and overlapping with an Ets-type factor. The
identification of Ets as a transcription factor in pituitary FGFR4
regulation was interesting, because the pituitary is well known to
express Ets as an important regulator of hormone gene expression
(20).
The PRL promoter FGF response element (FRE) has been mapped to two Ets
binding sites, and co-transfection of dn-Ets inhibits FGF activation
(20). That MAPK is upstream of Ets in this signaling cascade is corroborated by our demonstration that dnEts abrogates the
PRL response to FGF stimulation (Fig. 4). One of the FRE sites co-localizes with an Ets/Pit-1 composite. We have previously shown through co-transfection and mutational analysis that Ik1 and Ets1 functionally interact in regulating basal FGFR4 promoter activity (24). It was, therefore, particularly relevant to
determine whether the Ets/Ik composite is important in determining
pituitary FGFR4 regulation in response to FGF stimulation. Despite the
effect of Ets on basal and FGF-stimulated PRL promoter activity, the introduction of Ets induced basal FGFR4 promoter activity but was not
sufficient to confer an effect for FGF-1 or FGF-19 on the FGFR4
promoter, as shown schematically in Fig.
5. These data are in agreement with a
pivotal role for Ets in sorting MAPK-generated signals. In this
instance, Ets appears to sort and integrate distinct FGF-induced MAPK
signals through interaction with Pit-1 to the PRL promoter. In
contrast, the functional interaction between Ets and Ik does not appear
to be sufficient to confer a similar effect of FGF-19 onto the FGFR4
promoter.
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ACKNOWLEDGEMENTS |
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This work was supported by the Canadian Institutes of Health Research Grant MT-14404 to S. Ezzat and S. L. Asa and by Toronto Medical Laboratories.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. L. Asa, Univ. Health Network, 610 Univ. Ave. 4-302, Toronto, Ontario, Canada M5G 2M9 (E-mail: sylvia.asa{at}uhn.on.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 7, 2002;10.1152/ajpendo.00166.2002
Received 19 April 2002; accepted in final form 4 May 2002.
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