AJP-Endo Articles in PresS. Published on May 7, 2002 as DOI 10.1152/ajpendo.00166.2002 Yu et al, Page 1
Fibroblast Growth Factor Receptor 4 (FGFR4) Mediates Signalling to the Prolactin but Not FGFR4 Promoter
ShunJiang Yu1, Lei Zheng1, Sylvia L. Asa2, and Shereen Ezzat1
1
2
Department of Medicine, Mount Sinai Hospital and University of Toronto, Department of Pathology, University Health Network and University of Toronto The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
Running Title: Effect of FGF19 on the pituitary
Correspondence addressed to:
Dr. Sylvia L. Asa University Health Network 610 University Ave. 4-302 Toronto, Ontario Canada M5G 2M9 Phone: (416) 946-2099 Fax: (416) 946-6579 E.mail:
[email protected]
Copyright 2002 by the American Physiological Society.
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ABSTRACT. 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 PRL but not GH 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 FGF19-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.
Key Words: Fibroblast growth factor receptor 4, FGF-19, pituitary, ikaros, Ets1.
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INTRODUCTION The pituitary is the site of synthesis and the target of fibroblast growth factors (FGFs) that modulate hormone production and cell proliferation (1). FGF-2 (bFGF) was originally identified in bovine pituitary and is over-expressed in pituitary tumors, with the highest levels noted in aggressive tumors (2;3). FGF-4 has also been found in transforming DNA sequences from human prolactin (PRL)-secreting pituitary tumors (4). FGF ligands have also been shown to stimulate PRL secretion (1). Little is known, however, about the mechanism of this effect and the receptors involved in mediating it. FGF signalling is potentially mediated through one of four FGFRs, a complex family of transmembrane receptor tyrosine kinases (RTKs) (5). Each prototypic receptor is composed of 3 immunoglobulin (Ig)-like extracellular domains, 2 of which are involved in ligand binding, a single transmembrane domain, a split tyrosine kinase, and a COOH-terminal tail with multiple autophosphorylation sites (5). We have recently shown that targeted expression of a truncated pituitary tumor-derived FGFR4 (ptd-FGFR4) to the pituitary results in tumor formation (6). 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 (6). In the current study we sought to examine other functions of full length FGFR4 in the pituitary. While 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 (7) 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 signalling 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
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functional nuclear target that sorts and integrates distinct FGFR4-generated MAPK signals to the PRL and FGFR4 promoters.
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MATERIALS & METHODS
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, Inc.) with high glucose supplemented with 10% fetal bovine serum (FBS) (Sigma, Oakville, ON), 2 mM glutamine, 100 IU/ml penicillin and 100 mg/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 trans-sphenoidal pituitary surgery as previously described (8).
Plasmids. Promoter analysis of the human FGFR4 gene was performed as previously described (9). Fulllength human FGFR4 (6) and soluble dominant negative FGFR4 (sFGFR4)(10) were used as previously described. The orientation and sequence of all constructs was verified by restriction analysis and nucleotide sequencing. The expression vectors pSG5-Ets-1 encoding the chicken Ets-1 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) (11). PRL and GH promoter activity was analyzed with reporter constructs pSV2A-rPRL-luc containing the 422-bp fragment of the 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-b-Galactoside control vector
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(Promega, Madison WI) were included with each transfection. The results were normalized to bgalactosidase 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 x 105 cells per well), transfected the following day with 3 ml or 5 ml/well of lipofectamine and 1 or 2 mg of DNA per well as indicated. The total amount of transfected DNA was kept constant by adding empty vector. Transfection efficiency was monitored by simultaneous co-transfection with a b-galactosidase control expression plasmid CMV-bgal (20 ng/well). Forty-eight hours following 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 seconds in a luminometer. b-galactosidase activity was measured to normalize for variations in transfection efficiency. Promoter activity of each construct was expressed as firefly luciferase/bgal 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 x 104 cells/well), pre-incubated for 48 hrs in serum-free defined media (insulin [5 (:g/ml], transferrin [5 (:g/ml]). Treatments were based on time-(up to 48 hrs) and dose-(up to 100 ng/ml) response studies. Time and dose peak effects were utilized for further response studies
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using FGF-1 (Sigma, 50 ng/ml) or FGF-19 (Dr. A. L. Gurney, Genentech Inc., San Francisco, CA ; 50 ng/ml) and 10 U/ml of heparin was also carried out in serum free medium for 24 hrs at 37 C.
Messenger RNA analysis. Total RNA was extracted by the guanidinium isothiocyanate method. One mg of DNase-treated RNA was used for reverse transcription. This was performed using 2.5 U/ml of Murine Leukemia Virus reverse transcriptase, 2.5 mM MgCl2, 1 mM dNTP, 2.5 mM random hexamers, and 1 Unit/ml of RNase inhibitor. The integrity of RNA from each sample was assessed by amplification of the PGK-1 housekeeping gene as previously described (12). 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 #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 mg 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 55ºC with the rat PRL or GAPDH cDNA. Blots were washed and autoradiographed for 24-48 hrs.
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Western blot analysis. Protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein (50 mg) from cell lysates or media were solubilized in 2.5 x 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 rat PRL or GH (donated by the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD, 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 Histagged-sFGFR4 expression. Total and phosphorylated MAPK were 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 denistometric analysis.
Statistical Analyses. Data are expressed as mean + SEM. Differences were examined by one-way ANOVA or student’s t-test both with significance level of < 0.05.
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RESULTS Expression of FGF-19. Although FGFR4 is expressed by pituitary GH4C1 cells (9), we were not able to detect significant amounts of expression of its selective ligand (7) FGF-19 in the pituitary. Fig. 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 (7), the human colon adenocarcinoma cell line SW480 displayed markedly elevated levels of FGF-19 message (Fig. 1).
FGF-19-mediated FGFR4 activation induces PRL expression. We took advantage of the FGFR4 selective ligand FGF-19 to determine if it can activate FGFR4 and stimulate pituitary PRL expression. Since 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. Fig. 2a depicts western blotting analysis of lysates from GH4 cells exposed to the non-FGFR selective FGF-1 (13) 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 (9) 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 treatments (data not shown). As MAPK is considered to be an important mediator of FGF signalling (14), we examined whether FGF-1 and FGF-19 can similarly activate MAPK in wild type and FGFR4-
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transfected HEK 293 cells. We chose HEK 293 cells for this experiment as we had previously shown that these cells do not express significant amounts of FGFR1-3 (6). Both ligands induced phosphorylation of MAPK (Fig. 3b,c). Moreover, co-transfection of a soluble dominant negative FGFR4 isoform lacking the transmembrane and cytoplasmic domains (10) attenuated FGF-19 as well as FGF-1-induced MAPK activation (Fig. 3b,c). In contrast to the MAPK response to FGF-19 in FGFR4-transfected HEK 293 cells, untransfected GH4 cells demonstrated a more robust response to FGF-1 than FGF-19 (Fig. 3a). These findings are consistent with the high selectivity of FGF-19 for FGFR4 as compared to FGF-1 which is also capable of activating FGFR1-3 that are co-expressed in the pituitary (15).
FGFR4 Transduces Signalling to the PRL but not 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 (11). Fig. 4a demonstrates that FGF-19 can activate the PRL promoter but not influence its own receptor FGFR4. Treatment of GH4 cells using the non-FGFR4 selective ligand FGF-1 resulted in induction of PRL promoter activity by nearly 8-fold. As noted at the level of mRNA, the highly FGFR4selective ligand FGF-19 resulted in a more modest 2-fold 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 (15). Neither FGF-19 nor FGF-1 had influence on FGFR4 promoter activity however (Fig. 4a; right panel).
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Ets binding sites contribute to PRL and FGFR4 basal promoter activity. We have previously shown that over-expression of Ets1 results in activation of the FGFR4 promoter, an effect that was attenuated with disruption of the overlapping Ikaros (Ik) binding site (9). 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 (9). 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 dominant negative (dn) form of Ets on the FGFR4 as well as PRL promoter. Fig. 4b reveals that Ets1 transfection can activate either promoter by ~ 300% while dn-Ets inhibits both promoters compared with vector control-transfected GH4 cells.
FGF-19 and FGF-1 Enhance Ets1 Signalling to the PRL but not FGFR4 promoter Given the importance of MAPK in FGFR4 signalling 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. 4). Treatment with FGF-19 as well as FGF-1 resulted in further enhancement (2 and 6fold respectively) of Ets1-mediated activation of the PRL promoter (Fig. 4c; left panel). 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. 3c; right panel). 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
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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 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 plays 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 biologic 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 (16;17). 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 (18;19). Although the prototypic receptor has 3 Ig-like domains, FGFR isoforms that lack the first Ig-like domain are still efficiently activated by FGFs (20); 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 with loop 3; FGF-7 binds to loop 3 but not 2. FGF-7 binds to the loop 3 IIIb variant whereas FGF-2 binds the IIIc variant. FGF-1 binds both variants equally (13). FGF-19, however, uniquely binds intact FGFR4 (7). 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 signalling proteins, namely phospholipase C-g (PLC-g) (5;21). A 28 amino acid peptide containing Tyr-766 of FGFR1 has
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been identified as the major binding site for PLC-g; the analogous residue is conserved in all FGFRs with Tyr-754 being the putative site in FGFR4. Activated FGFR1 and FGFR4 homodimers interact with PLC-g (22). DNA synthesis and cell proliferation are equally induced by FGFR1 and FGFR4 (22). Mutations in the activation loop of kinase domains of FGFR1, FGFR3, and FGFR4 have revealed that all 3 receptor domains can equally transform NIH 3T3 cells, induce neurite outgrowth in PC12 cells, and phosphorylate Shp2, PLC-gamma, and MAPK (14). 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 (11), however, the role of FGFR4 in mediating this response was not identified and indeed little is known about which FGF/FGFR interactions are involved in mediating FGF signalling 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 (11). We thus sought to determine if 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 (7) can activate MAPK and the PRL promoter, thus defining a contribution for FGFR4 in mediating pituitary FGF signalling to the PRL promoter. As anticipated, PRL stimulation elicited by FGF-19 was not as robust as that induced by the nonFGFR selective FGF-1 ligand, consistent with FGF-1 mediated effects through multiple FGFRs that are expressed in the pituitary (15). The only other previously known function of FGF-19 has been a synergistic interaction with Wnt-8c in initiating inner ear development (23). 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 down-regulated in response to FGF ligand stimulation (24).
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In our analysis of the FGFR4 promoter we characterized a 214 bp of the 5’ region that is important for activity. Further mapping identified an approximately 40bp fragment (-64/-25) which demonstrated strong binding with nuclear extracts from pituitary GH4 cells (9). This fragment contained functional binding sites for the zinc finger-containing transcription factor Ikaros (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, since the pituitary is well known to express Ets as an important regulator of hormone gene expression (11). The PRL promoter FGF response element (FRE) has been mapped to two Ets binding sites and co-transfection of dn-Ets inhibits FGF activation (11). That MAPK is upstream of Ets in this signalling cascade is corroborated by our demonstration that dnEts abrogates the PRL response to FGF stimulation (figure 4). One of the FRE sites co-localizes with an Ets/Pit1 composite. We have previously shown through co-transfection and mutational analysis that Ik1 and Ets1 functionally interact in regulating basal FGFR4 promoter activity (9). It was, therefore, particularly relevant to determine if the Ets/Ik composite is important in determining pituitary FGFR4 regulation in response to FGF stimulation. Despite the effect of Ets on basal and FGFstimulated 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 figure 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 FGFinduced MAPK signals through interaction with Pit1 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 on to the FGFR4 promoter.
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Acknowledgements This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant MT-14404 to SE and SLA) and Toronto Medical Laboratories.
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References 1. Ezzat S The role of hormones, growth factors and their receptors in pituitary tumorigenesis. Brain Pathol 11:356-370, 2001. 2. Ezzat S, Smyth HS, Ramyar L, Asa SL Heterogeneous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 80:878-884, 1995. 3. Ezzat S, Horvath E, Kovacs K, Smyth HS, Singer W, Asa SL Basic fibroblast growth factor expression by two prolactin and thyrotropin-producing pituitary adenomas. Endocrine Pathol 6:125-134, 1995. 4. Gonsky R, Herman V, Melmed S, Fagin J Transforming DNA sequences present in human prolactin-secreting pituitary tumors. Mol Endocrinol 5:1687-1695, 1991. 5. Givol D, Yayon A Complexity of FGF receptors: genetic basis for structural diversity and functional specificity. FASEB J 6:3362-3369, 1992. 6. Ezzat S, Zheng L, Zhu X-F, Wu GE, Asa SL Targeted expression of a human pituitary tumor-derived isoform of FGF receptor 4 recapitulates pituitary tumorigenesis. J Clin Invest 109:69-78, 2002. 7. Xie M-H, Holcomb I, Deuel B, Dowd P, Huang A, Vagts A, Foster J, Liang J, Brush J, Gu Q, Hillan K, Goddard A, Gurney AL FGF-19, a novel fibroblast growth factor with unique specificity for FGFR4. Cytokine 11:729-735, 1999. 8. Asa SL, Ramyar L, Murphy PR, Li AW, Ezzat S The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production. Mol Endocrinol 15:589-599, 2001. 9. Yu SJ, Asa SL, Ezzat S Fibroblast growth factor receptor 4 is a target for the zinc-finger transcription factor Ikaros in the pituitary. Mol Endocrinol, in press. 10. Ezzat S, Zheng L, Yu S, Asa SL A soluble dominant negative fibroblast growth factor receptor 4 isoform in human mcf-7 breast cancer cells. Biochem Biophys Res Commun 287:60-65, 2001. 11. Schweppe RE, Frzer-Abel AA, Gutierrez-Hartmann A, Bradford AP Functional components of fibroblast growth factor (FGF) signal transduction in pituitary cells. J Biol Chem 272:30852-30859, 1997. 12. LeRiche V, Asa SL, Ezzat S Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab 81:656-662, 1996.
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13. Ornitz DM, Zu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M Receptor specificity of the fibroblast growth factor family. J Biol Chem 271:15292-15297, 1996. 14. Hart KC, Robertson SC, Kanemitsu MY, Meyer AN, Tynan JA, Donoghue JA Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4. Oncogene 29:3309-3320, 2000. 15. Abbass SAA, Asa SL, Ezzat S Altered expression of fibroblast growth factor receptors in human pituitary adenomas. J Clin Endocrinol Metab 82:1160-1166, 1997. 16. Mason IJ The ins and outs of fibroblast growth factors. Cell 78:547-552, 1994. 17. Becker D, Meier CD, Herlyn M Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor. EMBO J 8:3685-3691, 1989. 18. Ron D, Reich R, Chedid M, Lengel C, Cohen OE, Chan AM, Neufeld G, Miki T, Tronick SR Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor. J Biol Chem 268:5388-5394, 1993. 19. Vainikka S, Partanen J, Bellosta P, Coulier F, Basilico C, Jay M, Alitalo K Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction. EMBO J 11:4273-4280, 1992. 20. Crumley G, Bellot F, Kaplow JM, Schlessinger J, Jaye M, Dionne CA High-affinity binding and activation of a truncated FGF receptor by both aFGF and bFGF. Oncogene 6:2255-2262, 1991. 21. Mohammadi M, Honegger AM, Rotin D, Fischer R, Bellot F, Li W, Dionne CA, Jaye M, Rubinstein M, Schlessinger J A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-1. Mol Cell Biol 11:5068-5078, 1991. 22. Vainikka S, Joukov V, Wennstrom S, Bergman M, Pelicci PG, Alitalo K Signal transduction by fibroblast growth factor receptor-4 (FGFR-4). Comparison with FGFR-1. J Biol Chem 269:18320-18326, 1994. 23. Ladher RK, Anakwe KU, Gurney AL, Schoenwolf GC, Francis-West PH Identification of synergistic signals initiating inner ear development. Science 290:19651967, 2000. 24. Ali J, Mansukhani A, Basilico C Fibroblast growth factor receptors 1 and 2 are differentially regulated in murine embryonal carcinoma cells and in response to fibroblast growth factor-4. J Cell Physiol 165:438-448, 1995.
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Figure legends:
Figure 1. FGF-19 mRNA expression. Total RNA extracted from human pituitary adenomas (lanes 1-8), GH4 pituitary cells and the human colon carcinoma cell line SW480 was reverse transcribed (RT). The integrity of RNA and the efficiency of the RT reaction were first confirmed by PCR for the housekeeping gene PGK-1 (lower panel). Negative controls omitted reverse transcriptase (-RT) or replaced template with water. The size of PCR products was determined by comparison with concurrently electrophoresed standards (Kb) and the identity of all PCR products was confirmed by sequencing. FGF-19 mRNA was identified in the human colon adenocarcinoma SW480 cell line but not in pituitary.
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Figure 2. Effect of FGF-19 on Prolactin expression. (a) GH4 cells were transiently transfected with empty vector (pcDNA; left) or full length human FGFR4 (right) and treated for 24 hrs with FGF-1 or recombinant human FGF-19 (50 ng/ml) in the presence of added heparin (10 U/ml). Total cell lysates were subjected to western blotting with an antibody that recognizes rat PRL (upper) or actin (lower panel). Note stimulation by FGF-19 on PRL expression an effect that was further enhanced by FGFR4 transfection as evidence by increased basal as well as FGF-19-induced stimulation. (b) The densitometric analysis of mean values from 3 independent experiments is shown. (c) Northern blotting hybridization of RNA from GH4 cells treated as above with FGF-1 or FGF-19 with rPRL (upper panel) or GAPDH (lower panel) cDNA reveal induction of PRL gene expression that is more pronounced by FGF-1 (5-fold) than FGF-19 (2fold) as assessed by densitometry.
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Figure 3. FGF-19 Induces MAPK activation and is negatively influenced by sFGFR4. (a) GH4 cells were treated for 24 hrs with FGF-1 or FGF-19 (50 ng/ml) in the presence of added heparin (10 U/ml). Total cell lysates were subjected to western blotting with an antibody that recognizes phospho-specific (upper) or total MAPK (lower panel). Note the more robust stimulation by FGF-1 than FGF-19 on MAPK activation. (b) Untransfected HEK 293 cells (first two lanes) or transiently transfected HEK 293 cells with FGFR4 and treated 24 hrs later with FGF-1 or FGF-19 in the presence or absence of co-transfection with a soluble dominant negative FGFR4 isoform (sFGFR4) as indicated. Note that unlike in GH4 cells which express multiple types of FGFRs which can bind FGF-1, transfection of FGFR4 alone results in more comparable effects by FGF-1 and FGF-19 on MAPK phosphorylation. The co-transfection of sFGFR4 abrogates FGF-19-induced MAPK activation. Total MAPK levels, shown immediately below in the lower panel, are unaffected by treatments. (c) The densitometric analysis of mean values from 3 independent experiments is depicted in this bar graph.
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Figure 4. Comparison of PRL and FGFR4 promoter regulation by FGF-19 and FGF-1. (a) GH4 cells were stimulated with FGF-1 or FGF-19 after transfection with PRL (-422)- Luc promoter (left) or FGFR4 P(-115/ +99)-Luc minimal promoter (right). Cells were incubated for 24 hours before harvesting for luciferase and ß-gal assays. Note that FGF-19 and FGF-1 induce PRL but not FGFR4 promoter activity. (b) GH4 cells were transiently co-transfected either with the minimal FGFR4 promoter construct P(-115/ +99)-Luc or PRL (-422)- Luc as indicated along with equal amounts of the expression vector Ets1, or its corresponding empty vector control, or pAprEtsZ-neo (encoding dominant negative (dn) Ets) or its corresponding empty control vector and 20 ng of pCMVßgal to control for transfection efficiency. (c) GH4 cells co-transfected with the PRL or FGFR4 promoter and Ets1 or dnEts were treated with FGF-19 or FGF-1 as described above. Note that FGF19 and FGF-1 treatment further enhance Ets1-mediated activation of the PRL an effect which was attenuated by dnEts. Unlike the effects on PRL, neither FGF ligands could appreciably influence FGFR4 promoter activity. All results are presented as the mean + standard deviation (SD) of three separate experiments each performed in triplicate.
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Figure 5. Schematic illustration of proposed sorting of MAPK signals by Ets Partnerships. The PRL and FGFR4 promoters contain two key functional Ets binding sites. In the case of the PRL promoter, a composite Ets/Pit-1 element confers FGF responses and another downstream site co-localizes with basal transcription. Similarly, the FGFR4 promoter also contains two distinct Ets binding sites with a functional composite element that binds Ets and the zinc finger transcription factor Ikaros (Ik) as shown. Even though FGF-1 and FGF-19 signaling converge at MAPK (ERK1/2), these pathways are likely to target distinct Ets factors and their transcriptional partners. In the case of FGFR4, the Ets/Ik-1 functional partnership appears to sort distinct basal and FGF-stimulated signaling pathways to this receptor's promoter. This model provides a
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mechanism by which the combination of distinct Ets partnerships and their binding sites transduce specific growth factor responses.
