Differentiation of Lactotrope Precursor GHFT Cells in Response to Fibroblast Growth Factor-2
2000; Elsevier BV; Volume: 275; Issue: 28 Linguagem: Inglês
10.1074/jbc.m002129200
ISSN1083-351X
AutoresJudith López‐Fernández, Daniela Palacios, Ana I. Castillo, Rosa M. Tolón, Ana Aranda, Michael Karin,
Tópico(s)Pancreatic function and diabetes
ResumoThe mechanisms that control the emergence of different anterior pituitary cells from a common stem cell population are largely unknown. The immortalized GHFT cells derived from targeted expression of SV40 T antigen to mouse pituitary display characteristics of somatolactotropic progenitors in that they express the transcription factor GHF-1 (Pit-1) but not growth hormone (GH) or prolactin (PRL). We searched for factors capable of inducing lactotropic differentiation of GHFT cells. PRL gene expression was not observed in cells subjected to a variety of stimuli, which induce PRL gene expression in mature lactotropes. Only fibroblast growth factor-2 (FGF-2) was able to initiate the transcription, synthesis, and release of PRL in GHFT cells. However, induction of PRL expression was incomplete in FGF-2-treated cells, suggesting that additional factors are necessary to attain high levels of PRLtranscription in fully differentiated lactotropes. We also show that the FGF-2 response element is located in the proximal PRLpromoter. Stimulation of PRL expression by FGF-2 requires endogenous Ets factors and these factors as well as GHF-1 are expressed at low levels in the committed precursor, suggesting that these low levels are limiting for full PRL expression. Moreover, FGF-2 effect on lactotrope differentiation is mediated, at least partially, by stimulation of the Ras-signaling pathway. Our results suggest that, indeed, GHFT cells represent a valid model for studying lactotropic differentiation and that FGF-2 could play a key role both in initiating lactotrope differentiation and maintainingPRL expression. The mechanisms that control the emergence of different anterior pituitary cells from a common stem cell population are largely unknown. The immortalized GHFT cells derived from targeted expression of SV40 T antigen to mouse pituitary display characteristics of somatolactotropic progenitors in that they express the transcription factor GHF-1 (Pit-1) but not growth hormone (GH) or prolactin (PRL). We searched for factors capable of inducing lactotropic differentiation of GHFT cells. PRL gene expression was not observed in cells subjected to a variety of stimuli, which induce PRL gene expression in mature lactotropes. Only fibroblast growth factor-2 (FGF-2) was able to initiate the transcription, synthesis, and release of PRL in GHFT cells. However, induction of PRL expression was incomplete in FGF-2-treated cells, suggesting that additional factors are necessary to attain high levels of PRLtranscription in fully differentiated lactotropes. We also show that the FGF-2 response element is located in the proximal PRLpromoter. Stimulation of PRL expression by FGF-2 requires endogenous Ets factors and these factors as well as GHF-1 are expressed at low levels in the committed precursor, suggesting that these low levels are limiting for full PRL expression. Moreover, FGF-2 effect on lactotrope differentiation is mediated, at least partially, by stimulation of the Ras-signaling pathway. Our results suggest that, indeed, GHFT cells represent a valid model for studying lactotropic differentiation and that FGF-2 could play a key role both in initiating lactotrope differentiation and maintainingPRL expression. growth hormone prolactin radioimmunoassay fibroblast growth factor nerve growth factor base pair(s) reverse transcriptase polymerase chain reaction 1,4-piperazinediethanesulfonic acid RNase protection assay immunoreactive prolactin The anterior pituitary gland represents an excellent model system for studying selective gene activation. During embryonic development, different types of hormone producing cells that are highly specialized and synthesize distinct peptide hormones are sequentially derived from a common progenitor cell population within the anterior pituitary anlagen, Rathke's pouch (1.Dasen J.S. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 566-574Crossref PubMed Scopus (57) Google Scholar). Somatotropes, which express growth hormone (GH),1 and lactotropes, which express prolactin (PRL), are thought to be derived from a common precursor, the somatolactotrope (2.Theill L.E. Karin M. Endocr. Rev. 1993; 14: 670-689PubMed Google Scholar, 3.Hoeffler J.P. Boockfor F.R. Frawley L.S. Endocrinology. 1985; 117: 187-195Crossref PubMed Scopus (151) Google Scholar). The homeodomain transcription factor GHF-1/Pit-1 (4.Bodner M. Karin M. Cell. 1987; 50: 267-275Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 5.Bodner M. Castrillo J.L. Theill L.E. Deerinck Ellisman M. Karin M. Cell. 1988; 55: 505-518Abstract Full Text PDF PubMed Scopus (636) Google Scholar, 6.Ingraham H.I. Chen R. Mangalam H.J. Elsholtz H.P. Flynn S.F. Lin C.R. Simmons D.M. Swanson L. Rosenfeld G.M. Cell. 1988; 55: 519-529Abstract Full Text PDF PubMed Scopus (795) Google Scholar) is required both for GH and prolactin PRL gene activation and for emergence and expansion of both somatotropes and lactotropes (7.Li S. Creshaw E.B. Rawson E.J. Simmons D.M. Swanson L.W. Rosenfeld M.G. Nature. 1990; 347: 528-533Crossref PubMed Scopus (1051) Google Scholar, 8.Castrillo J.L. Theill L. Karin M. Science. 1991; 243: 814-817Crossref Scopus (88) Google Scholar). GHF-1 transcripts are detected several days before the emergence of GH- or PRL-producing cells (9.Dollé P. Castrillo J.L. Theill L.E. Deerinck T. Ellisman M. Karin M. Cell. 1990; 60: 809-820Abstract Full Text PDF PubMed Scopus (179) Google Scholar), suggesting the existence of a precursor cell for the somatolactotropic lineage. Using the 5′ GHF-1 regulatory region to target the immortalizing oncoprotein SV40 T-antigen in transgenic mice has immortalized this cell type. Mice expressing this transgene exhibit dramatic dwarfism and develop pituitary tumors, which express high levels of GHF-1 transcripts, low levels of GHF-1 protein, and no GH or PRL (10.Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (117) Google Scholar). This expression pattern is consistent with that of GHF-1-expressing progenitors detected between embryonic days 13 and 15 in the mouse (9.Dollé P. Castrillo J.L. Theill L.E. Deerinck T. Ellisman M. Karin M. Cell. 1990; 60: 809-820Abstract Full Text PDF PubMed Scopus (179) Google Scholar). A cell strain, designated GHFT, was established from these tumors. GHFT cells continue to exhibit the same phenotype as the original tumor and were therefore proposed to represent immortalized somatotrope/lactotrope progenitor (10.Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (117) Google Scholar). Thus, GHFT cells may constitute a convenient ex vivo system to study the mechanism of cell differentiation in an endocrine gland that itself is rather inaccessible to experimental manipulation during embryogenesis. The aim of this work was to identify factors that can induce the lactotropic differentiation of this committed precursor and explore their mechanism of action. Our efforts were focused on those agents that are known to stimulate PRL gene expression in differentiated lactotropes. Multiple hormones, growth factors, and oncogenes act in conjunction with GHF-1 to regulate pituitary-specific expression of the PRL gene. Those factors include ligands for nuclear hormone receptors (11.Simmons D.M. Voss J.W. Ingraham H.A. Holloway J.M. Broide R.S. Rosenfeld M.G. Swanson L.W. Genes Dev. 1990; 4: 695-711Crossref PubMed Scopus (571) Google Scholar, 12.Castillo A.I. Tolón R. Jimenez-Lara A.M. Aranda A. Mol. Endocrinol. 1999; 13: 1141-1154Crossref PubMed Scopus (49) Google Scholar), hypophysiotropic peptides that activate the protein kinase A or protein kinase C pathways (13.Maurer R.A. Nature. 1981; 294: 94-97Crossref PubMed Scopus (194) Google Scholar, 14.Day R.N. Maurer R.A. Mol. Endocrinol. 1989; 3: 3-9Crossref PubMed Scopus (90) Google Scholar, 15.Gourdji D. Laverriere J.N. Mol. Cell. Endocrinol. 1994; 100: 133-142Crossref PubMed Scopus (33) Google Scholar, 16.Howard P.W. Maurer R.A. J. Biol. Chem. 1995; 270: 20930-20936Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), or ligands of tyrosine kinase growth factor receptors (17.Jacob K.K. Stanley F.M. J. Biol. Chem. 1994; 269: 25515-25520Abstract Full Text PDF PubMed Google Scholar, 18.Pickett C.A. Gutierrez-Hartmann A. Mol. Cell. Biol. 1995; 15: 6777-6784Crossref PubMed Scopus (19) Google Scholar, 19.Ouyang L. Jacob K.K. Stanley F.M. J. Biol. Chem. 1996; 271: 10425-10428Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar). Among the latter, the family of fibroblast growth factors (FGFs) appears to play an important role in pituitary organogenesis (21.Treier M. Gleiberman A.S. O'Connell S.M. Szeto D.P. McMahon J.A. Rosenfeld M.G. Genes Dev. 1998; 12: 1691-1704Crossref PubMed Scopus (417) Google Scholar), in differentiation of lactotropes (22.Porter T.E. Wiles C.D. Frawley S. Endocrinology. 1994; 134: 164-168Crossref PubMed Scopus (36) Google Scholar), and recently in the dedifferentiation mechanism for lactotrope tumor pathogenesis (23.Shimon I. Huttner A. Said J. Spirina O.M. Melmed S. J. Clin. Invest. 1996; 97: 187-195Crossref PubMed Scopus (56) Google Scholar). Particularly, FGF-2 (or bFGF), which was originally isolated from the pituitary gland (24.Bohlen P. Baird A. Esch F. Ling N. Gospodarowicz D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5364-5368Crossref PubMed Scopus (182) Google Scholar, 25.Ferrara N. Scheigerer L. Neufeld G. Mitchell R. Gospodarowicz D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5773-5777Crossref PubMed Scopus (192) Google Scholar, 26.Li Y. Koga M. Kasayama S. Matsumotot K. Arita N. Hayakawa T. Sato B. J. Clin. Endocrinol. Metab. 1992; 75: 1436-1441Crossref PubMed Scopus (48) Google Scholar), stimulates PRL secretion from normal pituitary cells (27.Baird A. Mormede P. Ying Y. Wehrenberg P. Ueno N. Ling N. Guillemin R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5545-5549Crossref PubMed Scopus (141) Google Scholar) and from pituitary adenomas (28.Atkin S.L. Landolt A.M. Jeffreys R.V. Diver M. Radcliffe J. White M.C. J. Clin. Endocrinol. & Metab. 1993; 77: 831-837PubMed Google Scholar). FGF-2 was recently found to stimulate thePRL promoter in the lactotropic GH4 cell line, and the functional components of the signal transduction pathway activated by this growth factor have been determined (29.Schweppe R.E. Frazer-Abel A.A. Gutierrez-Hartmann A. Bradford A.P. J. Biol. Chem. 1997; 272: 30852-30859Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We report here that, among a variety of different agents tested, only FGF-2 was able to initiate the PRL gene transcription in GHFT cells. FGF-2 specifically stimulates PRL promoter activity in transient transfection assays in GHFT cells. The FGF-2 response element is located in the proximal promoter sequences, and Ets transcription factors are required for stimulation of thePRL promoter by FGF-2. GHFT cells express low levels of Ets factors, which could contribute to the reduced promoter responsiveness in these cells. In summary, our results indicate that FGF-2 is a strong up-regulator of PRL gene expression in somatolactotropic progenitors and that this factor is a strong candidate for a physiological inducer of lactotropic differentiation in vivoand probably also in maintaining the lactotropic phenotype of differentiated cells. GHFT cells were grown as described previously (10.Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (117) Google Scholar). Experiments were performed in a defined serum-free (Dulbecco's modified Eagle's medium-high glucose) medium without phenol red, containing insulin (10 μg/ml), sodium selenite (50 nm), human transferrin (10 μg/ml), ascorbic acid (10 μg/ml), 0.1% bovine serum albumin (fraction V), sodium pyruvate, glutamine, penicillin, and streptomycin. Cells were maintained at least overnight in this defined medium before the beginning of the experiments. GH4C1 and HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. For the experiments, the cultures were shifted to medium containing 10% AG1X8 resin-charcoal-stripped newborn calf serum and 24 h later shifted to serum-free medium. Treatments were administered in serum-free medium. Total RNA was isolated from cells as described previously (30.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). One μg of total RNA was used in RT-PCR reaction. The reverse transcription of RNA to cDNA (using cloned murine leukemia virus reverse transcriptase) and subsequent amplification (using GeneAmp® PCR process and AmpliTaq ® DNA polymerase) were performed all in a single reaction tube to avoid cross-contamination after first strand synthesis. RNA was copied to cDNA using random hexamers. To increase the specificity and sensitivity of PCR amplification, the “hot start” technique was used to suppress primer annealing to non-target sequences. AmpliWax™ PCR Gem 100 (Perkin-Elmer) was added to each single reaction tube containing a subset of amplification reagent for this proposal. For amplification of PRL cDNA, the specific primers 5′-CCCGAATACATCCTATCAAGAGCC-3′ and 5′-TTGATGGGCAATTTGGCACCTCAG-3′ were used. These primers amplified a fragment of 263 bp. As an internal control, the amplified cDNA fragment spanned two spliced exons, such that when genomic DNA was amplified the corresponding bands were larger due to the presence of an intron. PRL mRNA was detected by RPA. Total RNA from mouse pituitary was used as positive control. mRNA from GHFT and HeLa cells was isolated by Oligotex™ direct mRNA kit (Qiagen). The mouse PRL cDNA was inserted into pGEM2, and after XhoI linearization an antisense riboprobe was generated using SP6 RNA polymerase and [α-32P]UTP. The run-off transcription was allowed to proceed for 60 min at 37 °C. The reaction was stopped by digesting the DNA template with 10 units/μl DNase I for 15 min at 37 °C. The probe was purified from a polyacrylamide gel, eluted with the RNAid™ kit (Bio 101), and hybridization was performed overnight at 50 °C. The hybridization solution contained 80% formamide, 40 mm PIPES, pH 6.4, 1 mm EDTA, and 0.4 m NaCl. After hybridization, samples were digested using RNase-ONE™ (Promega; 50 units/sample, 50 min at 30 °C), precipitated with ethanol, and separated on a 6% polyacrylamide, 8 m urea gel. Autoradiography of the RPA showed a double protected fragment of 280 bp. Identical amounts of poly(A)+ RNA (16 μg) of each experimental group were used, except for total mouse pituitary RNA, that served as a positive control, where 0.5 and 2 μg of total RNA were used. RIA for mouse PRL was performed in duplicate as described previously (31.Escalada J. Cacicedo L. Ortego J. Melian E. Sanchez-Franco F. Endocrinology. 1996; 137: 631-637Crossref PubMed Scopus (33) Google Scholar). RIA components were purchased to Dr. Parlow (Pituitary Hormones and Cancer Center, Harbor-UCLA Medical Center). Iodination of mPRL with 125I was conducted using the chloramine-T method. Rabbit anti-mouse PRL serum (anti-mPRL AFP-131078) was used at a final dilution of 1/200,000 and samples were incubated for 18–24 h at room temperature prior to addition of secondary antibody. Medium samples were compared with a standard curve prepared with reference preparation (AFP-6476C), as described previously (31.Escalada J. Cacicedo L. Ortego J. Melian E. Sanchez-Franco F. Endocrinology. 1996; 137: 631-637Crossref PubMed Scopus (33) Google Scholar). The assay sensitivity was 0.48 ng/ml. After 48 of incubation with or without FGF-2, culture media (8 ml) from GHFT and HeLa cells were collected, frozen at −80 °C, lyophilized, and resuspended in 100 μl of phosphate buffer to load directly into the RIA. Constructs containing different fragments of the rat PRL promoter fused to luciferase or chloramphenicol acetyltransferase were described previously (12.Castillo A.I. Tolón R. Jimenez-Lara A.M. Aranda A. Mol. Endocrinol. 1999; 13: 1141-1154Crossref PubMed Scopus (49) Google Scholar, 20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar, 33.Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (419) Google Scholar). Expression vectors for GHF-1, c-Ets-1, dominant negative Ets-1 (encoding the DNA binding domain of c-Ets-2), oncogenic Ha-ras Val-12, and the dominant inhibitory Ha-ras Asn-17 mutant (20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar) were also used in the transfection assays. Cells were transfected with calcium phosphate and chloramphenicol acetyltransferase and luciferase activity determined as described previously (12.Castillo A.I. Tolón R. Jimenez-Lara A.M. Aranda A. Mol. Endocrinol. 1999; 13: 1141-1154Crossref PubMed Scopus (49) Google Scholar, 20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar). Reporter plasmids (1 μg/plate) were transfected alone or in combination with the amounts of expression vectors indicated in the corresponding figures. In all experiments the amount of DNA was kept constant by addition of the same amount of an “empty” expression vector. Assays were performed with nuclear extracts (32.Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19 (2499): 2499Crossref PubMed Scopus (2214) Google Scholar) from GHFT, GH4C1, and HeLa cells. The labeledPRL promoter fragment −176 to −101 was obtained by PCR using the oligonucleotides 5′-cccaagcttTGGCCACTATGTCTTCCT-3′ and 5′-CAATCATCTATTTCCGTCAT-3′ as primers. The first oligonucleotide was previously end-labeled with [32P]ATP using T4-polynucleotide kinase. For the binding reaction, the extracts were incubated on ice for 15 min in a buffer (20 mm Tris HCL (pH 7.5), 75 mm KCl, 1 mm dithiothreitol, 5 μg/μl bovine serum albumin, 13% glycerol) containing 3 μg of poly(dI-dC) and then for 15–20 min at room temperature with approximately 50,000 cpm of labeled DNA fragment. DNA-protein complexes were resolved on 6% polyacrylamide gels in 0.5× TBE buffer. The gels were then dried and autoradiographed at −70 °C. The levels of GHF-1 and Ets were determined by immunoblot analysis in GHFT, GH4C1, and HeLa cells. Cell extracts were prepared in a lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (32.Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19 (2499): 2499Crossref PubMed Scopus (2214) Google Scholar). Equal amounts of proteins (100 μg) were suspended in SDS sample buffer and resolved by 12% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane and, after blocking in 5% dried milk, were probed with a 1/1000 dilution of rabbit polyclonal antibody generated against GHF-1 (5.Bodner M. Castrillo J.L. Theill L.E. Deerinck Ellisman M. Karin M. Cell. 1988; 55: 505-518Abstract Full Text PDF PubMed Scopus (636) Google Scholar), and with 1/500 dilution of an antibody (sc112, Santa Cruz Biotechnology) that recognizes Ets-1 and Ets-2. Antigen-antibody complexes were detected by chemiluminescence. To identify extracellular factors capable of inducingPRL expression in GHFT precursor cells, we tested several hormones, peptides, and growth factors known to have a stimulatory effect on synthesis and/or release of PRL in differentiated lactotropes. We arbitrarily divided the factors into 3 groups. In group I, we analyzed the effects of ligands of nuclear hormone receptors that were demonstrated to transactivate the GHF-1 and/or thePRL genes, including vitamin D3 (12.Castillo A.I. Tolón R. Jimenez-Lara A.M. Aranda A. Mol. Endocrinol. 1999; 13: 1141-1154Crossref PubMed Scopus (49) Google Scholar, 33.Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (419) Google Scholar), retinoic acid (34.Rhodes S.J. Chen R. DiMattia G.E. Scully K.M. Kalla K.A. Lin S.-C., Yu, V.C. Rosenfeld M.G. Genes Dev. 1993; 7: 913-932Crossref PubMed Scopus (212) Google Scholar, 35.Sánchez-Pacheco A. Palomino T. Aranda A. Endocrinology. 1995; 136: 5391-5398Crossref PubMed Google Scholar), and both. 17β-Estradiol, a strong stimulator of PRL gene expression, was tested alone and in combination with retinoic acid and/or vitamin D3. In group II, we analyzed the peptides thyrotropin releasing hormone, epidermal growth factor, insulin, insulin-like growth factor-I, vasoactive intestinal peptide, and pituitary adenylate cyclase activating polypeptide 1–38. We also checked combinations of two, three, and four of these factors, along with combinations of group I substances. Group III included human nerve growth factor-β (NGF-β; Ref. 36.Missale C. Boroni F. Frassine M. Caruso A. Spano P. Endocrinology. 1995; 136: 1205-1213Crossref PubMed Google Scholar) and FGF-2 (22.Porter T.E. Wiles C.D. Frawley S. Endocrinology. 1994; 134: 164-168Crossref PubMed Scopus (36) Google Scholar), growth factors implicated in differentiation of cultured neonatal pituitary cells. We also checked these factors in combination with group I and group II agents. All treatments were performed under the same conditions for at least 48 h. After treatment, total RNA was isolated from cells, and expression of genes for PRL, GH, and GHF-1 was examined by RT-PCR. This screen revealed that only when FGF-2 was included in the experimental treatment, expression of PRL mRNA was detectable. None of the other agents either alone or in combination were able to induce PRL transcripts in GHFT cells (data not shown). Fig. 1 a shows a representative experiment of dose response of FGF-2 effect on GHFT cells. The expected amplified PRL band was obtained in cells treated with 10 nm FGF-2 for 24 h. This band was amplified when mouse pituitary RNA was used as a positive control but not when HeLa cell RNA was used as a negative control. Although 10 nm FGF-2 was the most effective dose in inducingPRL gene expression, incubation with 0.01 nmFGF-2 was sufficient to produce a weak detectable signal. Expression ofPRL mRNA was detectable within 6 h of treatment with FGF-2 and remained elevated for at least 30 h (Fig.1 b). The same treatment did not cause the appearance ofPRL mRNA in HeLa cells. Under all conditions at which FGF-2 induced PRL gene expression, no induction ofGH gene expression was detectable. However, GHFT cells continued to express GHF-1, a transcription factor necessary forGH and PRL gene expression (data not shown). To confirm that PRL gene expression was properly initiated after FGF-2 treatment, RPA was used (Fig.2 a). The expected protected double fragment corresponding to properly initiated PRL RNA was found in samples of FGF-treated GHFT cells (lane 6) but not in the untreated cells (lane 5). Longer exposures of the autoradiogram (up to 2 weeks) confirmed the results, demonstrating the presence of the double protected fragment ofPRL mRNA only in the positive control (total RNA from mouse pituitary) and in the newly differentiated precursor (GHFT cells after FGF-2 exposure) (Fig. 2 b). Therefore, RPA confirmed that FGF-2 was able to promote PRL expression. However, the levels of PRL transcripts produced by GHFT-treated cells were much lower than those expressed in mouse pituitary (lanes 3 and 4). These results confirm that, although FGF-2 appears to be an important factor for the initiation of PRL gene expression in GHFT cells and is able to initiate lactotrope differentiation, other factors are required to attain the high levels of PRL gene transcription found in the pituitary gland. After mRNA production, the next steps in expression of a polypeptidic hormone include translation, post-translational processing and secretion to the extracellular environment. Fig. 3 shows the effect of FGF-2 on PRL secretion by GHFT cells. Immunoreactive PRL (IR-mPRL) was essentially undetectable in medium from either untreated GHFT cells or from HeLa cells treated for different time periods with FGF-2 (Fig.3 a). However, following FGF-2 treatment, mPRL gradually accumulated in the culture medium and reached a level of 2 ng/ml after 24 h of treatment. This stimulatory effect was not lost after longer incubation intervals (72 and 120 h). As shown in Fig.3 b, treatment with a low dose of FGF-2 (0.1 nm) for 48 h was enough to produce detectable PRL secretion. Detection of PRL in the cell culture supernatants confirms that FGF-2 initiates differentiation of GHFT cells into PRL-expressing and secreting lactotropic cells. To analyze the elements that mediate increased PRL gene transcription in response to FGF-2 treatment, transient transfection experiments with reporter plasmids containing different fragments (Fig.4 a) of the rat PRLpromoter were performed. As shown in Fig. 4 b, incubation of GHFT cells with FGF-2 increased the activity of a promoter construct which contains the PRL distal enhancer (between −1.8 and 1.5 kilobase pairs) ligated to the −422/+34 PRL promoter fragment. In five independent experiments, incubation for 8–9 h with 1 nm FGF-2 increased luciferase activity by 2.7 ± 0.3-fold (p < 0.001). A 24-h incubation stimulated activity by 1.9 ± 0.2 (p < 0.01). A construct extending only to −422 bp, which does not contain the distal enhancer, was also stimulated by FGF-2 (Fig. 4 b). In contrast, a plasmid in which the −78/+34 promoter fragment was ligated to the distal enhancer was not significantly activated by FGF-2. The activity of the −38/+34 fragment was very low and was not affected by FGF-2. These data demonstrate that the elements responsible for FGF-2 responsiveness are contained between nucleotides −422 and −78. These results are in agreement with the previous observation that FGF-2 induction of the PRL promoter in GH4 cells maps to this region. A more detailed mapping was performed with plasmids extending to −176, −101, and −70 bp, and to better resolve the effect of FGF-2 the transfections were performed in GH4C1 cells, in which incubation with FGF-2 produced a stronger stimulation of PRL promoter activity (Fig. 4 c). A similar increase (9-fold) was found with constructs containing either the entire 5′-flanking region (3 kilobase pairs) or extending only to −176 bp. However, stimulation decreased to a mere 2-fold when sequences between −176 and −101 were deleted, and disappeared upon a deletion to −70. Thus, the region between −176 and −101 bp of the PRL promoter, which contains a GHF-1 binding site overlapping with an Ets binding site (37.Conrad K.E. Oberweter J.M Vaillancourt R. Johnson G.L. Gutierrez-Hartmann A. Mol. Cell. Biol. 1994; 14: 1553-1565Crossref PubMed Scopus (71) Google Scholar,38.Bradford A.P. Wasylyk C. Wasylik B. Gutierrez-Hartmann A. Mol. Cell. Biol. 1995; 15: 2849-2857Crossref PubMed Scopus (113) Google Scholar), significantly contributes to the induction of promoter activity by FGF-2. The role of the proximal Ets binding sites in the residual stimulation by FGF-2 of the reporter that extents −101 bp is demonstrated by the finding that the −101mut reporter in which the Ets binding sites were rendered non-functional (20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar) did not show a significant response to FGF-2 (Fig. 4 c). In different experiments, basal PRL promoter activity was found to be consistently lower in precursor GHFT cells than in the PRL-producing GH4C1 cells. As both GHF-1 and Ets factors appear to play an important role in PRL gene transcription, we tested the possibility that a lower expression of these transcription factors in the precursor cells could contribute to low promoter activity. Indeed, as analyzed by gel retardation assay with the prolactin promoter fragment −176 to −101, GHFT cells expressed lower levels of GHF-1 than GH4C1 cells (Fig.5 a). GHF-1 and Ets protein levels were then compared by Western blotting of GHFT, GH4C1, and HeLa cell extracts. This analysis confirmed the reduced content of GHF-1 in GHFT cells. The anti-GHF-1 antibody recognized the characteristic 31- and 33-kDa doublet in pituitary cells, which was less abundant in GHFT cells (Fig. 5 b). In the blot shown in the figure, obtained after a long exposure, two other weaker bands of 36 and 28 kDa were observed in GH4C1 cells, and no bands were detected in HeLa cells. In addition, the levels of endogenous Ets factors were markedly lower in GHFT cells than in GH4C1 or HeLa cells (Fig. 5 b). To functionally determine the role of these factors in basalPRL promoter activity as well as in its induction by FGF-2, we examined the influence of ectopically expressed c-Ets-1 alone or in combination with GHF-1 on PRL promoter activity. Overexpression of GHF-1 and/or c-Ets-1 did not further activate thePRL promoter in GH4C1 cells that already contain high endogenous levels of these factors (20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref PubMed Scopus (43) Google Scholar). However, cotransfection with the c-Ets-1 vector increased the activity of the PRLpromoter in GHFT cells and overexpression of GHF-1 further enhanced this activation (Fig. 5 c). After overexpression of c-Ets-1 and GHF-1, PRL promoter activity in GHFT cells was quite similar to that found in GH4C1 cells. These results suggest that the endogenous levels of GHF-1 and Ets factors are limiting in GHFT cells and confirm the important role of Ets proteins in activation of this promoter. Ras acts synergistically with Ets and GHF-1 to stimulate PRLpromoter activity in lactotropic cell lines (20.Castillo A.I. Tolon R.M. Aranda A. Oncogene. 1998; 16: 1981-1991Crossref
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