Pituitary Tumor AP-2α Recognizes a Cryptic Promoter in Intron 4 of Fibroblast Growth Factor Receptor 4

Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of proliferative functions, and FGFR4 is expressed differentially in normal and neoplastic pituitary. Human pituitary tumors express a truncated FGFR4 isoform (ptd-FGFR4) for which transcription is initiated from a downstream alternative site. Analysis of FGFR4 intronic sequences predicted a possible promoter within intron 4 (In4) including a classic TATA box with a possible transcriptional start site in intron 5. We show here that the human In4 sequence can direct luciferase reporter activity in transfected pituitary GH4 cells. Four overlapping fragments (A1, A2, B1, and B2) of this intron were examined by electromobility shift assay using nuclear extracts from rat pituitary tumors. Of these, fragment B2 formed complexes with nuclear rat pituitary GH4 extracts that were competed specifically by wild type but not mutant oligonucleotides for the neural crest cell lineage-derived activating transcription factor AP-2. Conversely, an AP-2 consensus sequence probe was competed by the In4 B2 oligonucleotide but not by other fragments of the same intron. The In4 B2 complex was competed partially by NFκB, supershifted by an AP-2α-specific antibody, and co-migrated with the same probe incubated with recombinant AP-2α protein. We also examined the ability of primary human pituitary tumor extracts to interact with the In4 B2 fragment. Pituitary tumor-In4 B2 complexes were competed specifically by wild type AP-2 but not mutant AP-2 oligonucleotides. Western blotting revealed higher levels of AP-2α expression in primary human pituitary tumors than in nontumorous tissue. Mutagenesis of the putative AP-2 binding site in In4 B2 resulted in a marked loss of promoter activity in a luciferase assay. AP-2α transfection in the presence of the histone deacetylase inhibitor trichostatin-A resulted in enhanced expression of endogenous ptd-FGFR4. These data indicate that a cryptic promoter within intron 4 binds AP-2α. AP-2α and chromatin changes may contribute to the utilization of an alternative transcription start site leading to the genesis of the tumorigenic ptd-FGFR4 isoform. Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of proliferative functions, and FGFR4 is expressed differentially in normal and neoplastic pituitary. Human pituitary tumors express a truncated FGFR4 isoform (ptd-FGFR4) for which transcription is initiated from a downstream alternative site. Analysis of FGFR4 intronic sequences predicted a possible promoter within intron 4 (In4) including a classic TATA box with a possible transcriptional start site in intron 5. We show here that the human In4 sequence can direct luciferase reporter activity in transfected pituitary GH4 cells. Four overlapping fragments (A1, A2, B1, and B2) of this intron were examined by electromobility shift assay using nuclear extracts from rat pituitary tumors. Of these, fragment B2 formed complexes with nuclear rat pituitary GH4 extracts that were competed specifically by wild type but not mutant oligonucleotides for the neural crest cell lineage-derived activating transcription factor AP-2. Conversely, an AP-2 consensus sequence probe was competed by the In4 B2 oligonucleotide but not by other fragments of the same intron. The In4 B2 complex was competed partially by NFκB, supershifted by an AP-2α-specific antibody, and co-migrated with the same probe incubated with recombinant AP-2α protein. We also examined the ability of primary human pituitary tumor extracts to interact with the In4 B2 fragment. Pituitary tumor-In4 B2 complexes were competed specifically by wild type AP-2 but not mutant AP-2 oligonucleotides. Western blotting revealed higher levels of AP-2α expression in primary human pituitary tumors than in nontumorous tissue. Mutagenesis of the putative AP-2 binding site in In4 B2 resulted in a marked loss of promoter activity in a luciferase assay. AP-2α transfection in the presence of the histone deacetylase inhibitor trichostatin-A resulted in enhanced expression of endogenous ptd-FGFR4. These data indicate that a cryptic promoter within intron 4 binds AP-2α. AP-2α and chromatin changes may contribute to the utilization of an alternative transcription start site leading to the genesis of the tumorigenic ptd-FGFR4 isoform. The pituitary gland is the site of synthesis and the target of several growth factors that modulate hormone production and cell proliferation (1Asa S.L. Ezzat S. Nat. Rev. Cancer. 2002; 2: 836-849Crossref PubMed Scopus (290) Google Scholar). Of these, members of the fibroblast growth factor (FGF) 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; ptd-FGFR4, human pituitary tumor-derived FGFR4; In4, intron 4; EMSA, electrophoretic mobility shift assay; B2ex, extended B2 fragment; Ik, Ikaros; GST, glutathione S-transferase; Luc, luciferase; STAT, signal transducers and activators of transcription. family have been shown to be overexpressed in human pituitary tumors (2Ezzat S. Smyth H.S. Ramyar L. Asa S.L. J. Clin. Endocrinol. Metab. 1995; 80: 878-884Crossref PubMed Google Scholar). FGF signaling is mediated through one of four FGF receptors (FGFRs), a complex family of transmembrane receptor tyrosine kinases (3Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (400) Google Scholar). Each prototypic receptor is composed of three Ig-like extracellular domains, a single transmembrane domain, a split tyrosine kinase, and a carboxyl-terminal tail with multiple autophosphorylation sites (3Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (400) Google Scholar). Multiple cell-bound or secreted forms of FGFR1, -2, and -3 have been characterized as resulting from alternative initiation sites, alternative splicing, exon switching, or variable polyadenylation. Recently, we found that pituitary tumors express a cytoplasmic form of FGFR4 lacking the signal peptide and the first two extracellular Ig-like domains (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar). Full-length FGFR4 has been reported to be expressed mainly in adult lung, liver, kidney, pancreas, muscle, and spleen (5Partanen J. Mäkelä T.P. Eerola E. Korhonen J. Hirvonen H. Claesson-Welsh L. Alitalo K. EMBO J. 1991; 10: 1347-1354Crossref PubMed Scopus (460) Google Scholar, 6Hughes S.E. J. Histochem. Cytochem. 1997; 45: 1005-1019Crossref PubMed Scopus (218) Google Scholar). It initially was considered not to have a significant role in tumorigenesis. However, FGFR4 has been shown to mediate membrane ruffling in breast carcinoma cells (7Johnston C.L. Cox H.C. Gomm J.J. Coombes R.C. Biochem. J. 1995; 306: 609-616Crossref PubMed Scopus (47) Google Scholar) and to modulate erythroid cell proliferation (8Koritschoner N.P. Bartunek P. Knespel S. Blendinger G. Zenke M. Oncogene. 1999; 18: 5904-5914Crossref PubMed Scopus (19) Google Scholar). Moreover, mutational analyses of the kinase domains of FGFR1, FGFR3, and FGFR4 revealed that all three receptor domains have comparable transforming properties (9Hart K.C. Robertson S.C. Kanemitsu M.Y. Meyer A.N. Tynan J.A. Donoghue J.A. Oncogene. 2000; 29: 3309-3320Crossref Scopus (224) Google Scholar). We have shown that targeted expression of a human pituitary tumor-derived FGFR4 isoform (ptd-FGFR4) recapitulates pituitary tumorigenesis (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar). This truncated form of FGFR4 is a result of transcription initiation in intron 5 and a translation start site in exon 6 (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar). In this report, we investigate an alternative FGFR4 promoter that may be implicated in the genesis of ptd-FGFR4. Sequence analysis of the human FGFR4 gene predicts a possible promoter within intron 4 including a classic TATA box with a possible transcription start site in intron 5 (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar, 10Becker M. Brauninger A. Wolf G. Kaufmann M. Strebhardt K. Biochem. Biophys. Res. Commun. 2000; 276: 493-501Crossref PubMed Scopus (9) Google Scholar). We thus focused on intron 4 and the surrounding region and determined the relevant cis-DNA-binding elements in pituitary cells that may contribute to utilization of this site. Cell Culture and Tissue—HEK 293 and the rat pituitary tumor-derived GH4 cell lines were grown in Dulbecco's modified Eagle's medium (Invitrogen) with high glucose supplemented with 10% fetal bovine serum (Sigma), 2 mm glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin and incubated at 37 °C with 5% CO2. 24 h before transfection, cells were plated with Dulbecco's modified Eagle's medium containing 10% serum. Primary human pituitary tumors were collected at the time of trans-sphenoidal pituitary surgery and snap frozen. Plasmids—Promoter analysis of the human FGFR4 gene was performed with the assistance of a gene finder. The convention for sequence coordinates with +1 as the first base of the coding sequence in exon 1 was adopted. Analysis of FGFR4 intronic sequences predicted a possible promoter within intron 4 (nucleotides 5056–5166) and a classic TATA box (nucleotides 5412–5417) with a possible transcription start site in intron 5 (nucleotide 5453). To generate the putative intron 4 (In4)-inclusive promoter, a KpnI/XhoI fragment of 313 bp consisting of intron 4 and exon 5 was derived by PCR from PAC# 32C5 (11Kostrzewa M. Muller U. Mamm. Genome. 1998; 9: 131-135Crossref PubMed Scopus (33) Google Scholar). The upstream primer was 5′-GGACCTCTCGAATAGGCACA-3′ (nucleotides 5021–5040), and the downstream primer was 5′-GGCTTACGGAGGTTACGCCA-3′ (nucleotides 5333–5314). After cloning into TA vector (Invitrogen), products were subcloned into their corresponding sites of the promoterless firefly luciferase expression vector pGL3 (Promega, Madison, WI) to yield the P313-Luc plasmid. The orientation and sequence of all constructs were verified by restriction analysis and nucleotide sequencing, respectively. The expression vector pcDNA-AP-2α encoding the complete sequence for AP-2α cloned into pcDNA3.1 (Invitrogen) KpnI/XhoI sites was used as described previously (12McPherson L.A. Weigel R.J. Nucleic Acids Res. 1999; 27: 4040-4049Crossref PubMed Scopus (96) Google Scholar). Site-directed Mutagenesis—Site-directed mutagenesis was performed using a transformer site-directed mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, CA) following the manufacturer's instructions. Mutations of the AP-2 transcription core binding site (-CCCTCCAGC-) substituted by -CTTTCCAGC- and the NFκB binding site (-GGGGCCTTCC-) substituted by -GGCGCCTTGC- were introduced separately into the P313-Luc construct and In4 B2 fragment using mutagenic primers for the sites. Another primer containing the mutation of a unique restriction site was used as a selection marker. Mutations were confirmed by restriction digestion and nucleotide sequencing. Transfection and Luciferase Assays—All plasmid reporters and expression vectors were prepared by column chromatography (QiaGen, Missisauga, Ontario) for sequencing and transfections. Cells were transfected by the LipofectAMINE method (Invitrogen) according to the manufacturer's protocol. Cells were plated into six-well cluster dishes (7 × 104 cells/well) and were transfected the following day with 3 or 5 μl/well LipofectAMINE and 1 or 2 μg of DNA/well as indicated. The total amount of transfected DNA was kept constant by adding empty vector. Transfection efficiency was monitored by simultaneous cotransfection with a β-galactosidase reporter construct (20 ng/well). Histone deacetylase inhibition was performed using trichostatin-A (Sigma) at 100 ng/ml for 24 h. 48 h following transfection, cells were lysed in a buffer containing 25 mm glycylglycine, 15 mm MgSO4, 4 mm EGTA, Triton X-100, and 1 mm dithiothreitol. Luciferase activity was measured for 20 s in a luminometer. β-Galactosidase activity was measured to normalize for variations in transfection efficiency. Promoter activity of each construct was expressed as firefly luciferase/β-galactosidase activity. Each experiment was performed independently on three separate occasions with at least triplicate wells in each experiment. Preparation of Nuclear Extracts—Nuclear extracts were prepared by washing cells in 1× phosphate-buffered saline and lysing in 100 μl of buffer (10 mm HEPES, pH 7.9, 1 mm dithiothreitol, 1 mm EDTA, 60 mm KCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride) for 5 min on ice. The pellet was resuspended in 100 μl of the nuclear resuspension buffer (0.25 mm Tris-HCl, pH 7.8, 60 mm KCl, 1 mm dithiothreitol, 1.5 mm phenylmethylsulfonyl fluoride) and lysed with three cycles of freezing and thawing to 37 °C. After centrifugation at 13,000 rpm for 10 min at 4 °C, the clear supernatant was collected and kept at -80 °C for further analysis. Protein concentrations were determined by the Bio-Rad protein assay. Electrophoretic Mobility Shift Assays (EMSAs)—Oligonucleotides were labeled with [γ-32P]ATP using the T4 DNA polynucleotide kinase. Five μg of nuclear protein extracts and 2 ng of labeled oligonucleotides were allowed to bind for 30 min at room temperature in a final volume of 20 μl of binding buffer (20 mm HEPES, pH 7.9, 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.5 mm MgCl2, 2% glycerol, and 1 μg of poly(dI-dC) (Amersham Biosciences, Peapack, NJ). Protein-DNA complexes were resolved in 4% polyacrylamide gels containing 0.5× Tris borate/EDTA. Overlapping double-stranded oligonucleotide fragments of intron 4 of FGFR4 (nucleotides 5056–5166) were used as probes and for competition in EMSAs as shown in Fig. 1 and detailed as follows: fragment A1 (nucleotides 5056–5085), 5′-GTCAGTAGGTCTCCAAGGACTTGTGTCCCG-3′; fragment A2 (nucleotides 5077–5115), 5′-TGTGTCCCGCTGCTGCTCATCTGATCACTGAGAAGAGGA-3′; fragment B1 (nucleotides 5106–5136), 5′-GAGAAGAGGAGGCCTGTGTGGGAACACACGG-3′; and fragment B2 (nucleotides 5127–5166), 5′-GAACACACGGTCATTCTAGGGGCCTTCCCCTGCCCTCCAG-3′. In some experiments, the B2 fragment was further extended (B2ex) to include the first six nucleotides of exon 5, thereby including the entire AP-2 consensus binding site, 5′-GAACACACGGTCATTCTAGGGGCCTTCCCCTGCCCTCCAGCACCCT-3′ (Fig. 1). Competitor double-stranded oligonucleotides containing transcription binding sites were as follows: AP-2, sense 5′-CCACAAACGACCGCCCGCGGGCGGT-3′ (commercially obtained from Geneka Biotech Inc., Carlsbad, CA) and its complementary strand; the mutant oligonucleotide for mAP-2, sense 5′-CCACAAACGACCGATTGCGGGCGGT-3′ and its complementary strand; AP-1, 5′-CGCTTGATGAGTCAGCCGGAA-3′ and its complementary strand; AP-2α, 5′-GATCGAACTGACCGCCCGCGGCCCGT-3′ and its complementary strand; AP-4, sense 5′-TTACTCCCAGCTCCAGCCGG-3′ and its complementary strand; and NFκB, sense 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and its complementary strand (synthesized by Sigma). Complementary strands were annealed in a buffer of 10 mm Tris-Cl, pH 8.0, 50 mm NaCl, and 1 mm EDTA before being used as probes or shift probes were with a column competition or 100 of fragment was as were on 4% polyacrylamide gels containing 0.5% Tris buffer and 2% were a and Western concentrations were determined by the Bio-Rad protein assay. of protein from cell or nuclear were in on polyacrylamide and to were incubated with the of AP-2α CA) or FGFR4 or a The of the was confirmed by of with Sequence analysis of the upstream fragments in intron 4 revealed putative binding sites for the transcription factors AP-2, NFκB, AP-4, and as in Fig. 4 of FGFR4 for which factors may interact with this putative intronic we using EMSAs were performed using nuclear extracts from rat pituitary tumor GH4 cells or from primary human pituitary tumors as described previously S. Asa S.L. Ezzat S. Endocrinol. 2002; PubMed Scopus Google Scholar). The overlapping fragments of bp of intron 4 that were as A2, B1, and These fragments were labeled and used as probes for the of Fig. an of an using GH4 rat pituitary tumor nuclear incubated with the In4 A1 We found of complexes that be competed by putative transcription factors using this In4 A1 fragment. were obtained with the In4 A2 and In4 B1 In of the same nuclear extracts with the In4 B2 fragment complexes that were competed specifically by wild type but not mutant AP-2 oligonucleotides (Fig. This complex was not competed by oligonucleotides for predicted binding sites for AP-4, Ik, or but was competed partially with an oligonucleotide for The putative AP-2 binding site is on the (Fig. 1). of the probe by 6 nucleotides into exon 5 to complete the AP-2 consensus binding site resulted in a complex that was supershifted by AP-2α (Fig. To further the of the putative intron 4 with AP-2, we examined the ability of an AP-2 consensus probe to form complexes with GH4 nuclear This complex was competed specifically by the In4 B2 fragment but not by other fragments from the same intron (Fig. Moreover, the In4 B2 probe formed complexes with the recombinant protein (Fig. of the putative AP-2 binding site in the B2 fragment resulted in a complex that was not competed by wild type AP-2 (Fig. with the presence of a single AP-2 binding site in this fragment of intron This was competed partially by NFκB (Fig. with an NFκB probe also formed complexes with GH4 nuclear extracts that were competed specifically by In4 B2 but not by the In4 A2 sequence (Fig. These indicate that the region of the cryptic promoter within In4 B2 consensus binding sites for AP-2 and GH4 cells express nuclear factors that bind to sites, and this complex is supershifted with to AP-2α, thus that AP-2α is one factor that the region In4 Primary Human Pituitary of a human pituitary tumor-derived FGFR4 we for a role of the AP-2 binding site in intron 4 in primary human pituitary tumors. Nuclear from primary human pituitary tumors formed the same complexes with the In4 B2 probe (Fig. These complexes were competed specifically by an oligonucleotide for wild type AP-2 but not by mutant AP-2. To that primary human pituitary tumors express the AP-2α we examined by Western blotting extracts from primary human pituitary characterized as the truncated ptd-FGFR4 (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar). We a protein of using an AP-2α-specific in nontumorous pituitary and in pituitary (Fig. This with protein derived from 293 cells transfected with an expression vector for AP-2α. for AP-2α protein was in the pituitary tumors than in nontumorous pituitary tissue. of AP-2 in of FGFR4 To the of the AP-2 binding site in intron 4 on promoter we examined the of mutation of this site on promoter activity. of the AP-2 site in the B2 fragment resulted in marked in luciferase activity (Fig. In mutation of the NFκB site also resulted in a loss of reporter activity but to a than that with the loss of the core AP-2 binding site. Conversely, of exon 5 and intron 5 not promoter activity not Transfection of AP-2α in GH4 cells with trichostatin-A resulted in enhanced expression of ptd-FGFR4 (Fig. that AP-2α in pituitary with chromatin changes as result in enhanced utilization of a cryptic promoter within intron to the genesis of the truncated receptor ptd-FGFR4. the of a tumor-derived form of FGFR4 in which transcription is initiated from an alternative downstream site (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google we to elements that can be implicated in the of a downstream cryptic the human and FGFR4 promoter and their transcription start sites M. Brauninger A. Wolf G. Kaufmann M. Strebhardt K. Biochem. Biophys. Res. Commun. 2000; 276: 493-501Crossref PubMed Scopus (9) Google Scholar, M. Muller U. Mamm. Genome. 1998; 9: 131-135Crossref PubMed Scopus (33) Google Scholar). of putative transcription factor binding or transcriptional activity. analysis of the FGFR4 promoter revealed a region bp upstream of the start site that we found to be for reporter activity in pituitary cells S. Asa S.L. Ezzat S. Endocrinol. 2002; PubMed Scopus Google Scholar). Overlapping fragments of this promoter were examined by complexes were with fragments that multiple binding sites for This was with the role of in the of other lacking TATA and sequence including H. H. S. Biochem. Biophys. Res. Commun. 1992; PubMed Scopus Google and A. K. Yayon A. D. Oncogene. 1992; Google Scholar). each of bp of sequence to the transcription initiation site to transcriptional activity to the J. 1998; PubMed Scopus Google Scholar). We further characterized fragment to of as binding with nuclear extracts from rat pituitary cells. This fragment predicted sites for binding the protein Ik, by two sites for and factors S. Asa S.L. Ezzat S. Endocrinol. 2002; PubMed Scopus Google Scholar). These to the of in the pituitary and to as an to FGFR4 transcriptional The to AP-2α as an factor that a downstream cryptic promoter in intron 4 of The family of transcription factors of four members as AP-2α, and (12McPherson L.A. Weigel R.J. Nucleic Acids Res. 1999; 27: 4040-4049Crossref PubMed Scopus (96) Google Scholar, M. J. 276: PubMed Scopus Google Scholar). The carboxyl-terminal of a and a that DNA binding and In has been that AP-2α is in cell proliferation through of a of for as as M. J. 276: PubMed Scopus Google Scholar, P. J. M. H. J. 2002; PubMed Scopus Google Scholar, L.A. Weigel R.J. J. 2002; PubMed Scopus Google Scholar). also to an role for NFκB in binding and intron In the NFκB has been implicated in a of including to inhibition by in pituitary cells M. A. L. L. P. Endocrinol. 2002; Google Scholar). of the R.J. J. 276: PubMed Scopus Google Scholar) and serum A1 J. 276: PubMed Scopus Google Scholar) gene also partially overlapping consensus sites for AP-2 and These a complex two transcription promoter analysis that NFκB and AP-2α in the of this cryptic The of two sites further a two factors to FGFR4 gene pituitary hormone including K. PubMed Scopus Google M. Endocrinol. PubMed Scopus Google the hormone receptor E. A. J. PubMed Scopus Google and receptors D. PubMed Scopus (47) Google have been described as containing AP-2 binding sites. The role of AP-2 in transcriptional of of putative target to be The presence of AP-2 binding sites in intronic sequences has been using intron 1 of the gene revealed at least two 1 and In 1 In 1 an binding site that with and fibroblast nuclear as as with recombinant AP-2α protein. shift experiments, that the nuclear factors from AP-2α but to the AP-2 family M. M. J. 1999; Google Scholar). Analysis of the receptor promoter revealed that the first intron activity. This region was shown to include a AP-2 that is by M. E. E. E. M. S. Oncogene. 2002; PubMed Scopus Google Scholar). for AP-2α as a factor that is expressed in pituitary is to the region of an intronic and is a in the of a cryptic FGFR4 The of in cell and tumorigenesis to be (1Asa S.L. Ezzat S. Nat. Rev. Cancer. 2002; 2: 836-849Crossref PubMed Scopus (290) Google Scholar). of a human pituitary tumor-derived truncated FGFR4 isoform with properties from wild type FGFR4 (4Ezzat S. Zheng L. Zhu X.F. Wu G.E. Asa S.L. J. Clin. Invest. 2002; 109: 69-78Crossref PubMed Scopus (149) Google Scholar) has a unique to the of alternative transcription initiation on FGFR4 on the of an intronic sequence of FGFR4 by AP-2α and the expression of this factor by pituitary tumors to the that may alternative promoter utilization in cells.

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