A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene
2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês
10.1074/jbc.m709502200
ISSN1083-351X
AutoresAmy L. Blount, Joan Vaughan, Wylie Vale, Louise M. Bilezikjian,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoFollistatins exert critical autocrine or paracrine control in many tissues by binding and bio-neutralizing activin and several other transforming growth factor-β ligands. In the pituitary, activin acts locally to induce follistatin expression and thus modulate its own actions. This local feedback loop safeguards against excessive activin signaling and maintains the necessary balance of activin and follistatin tone. To better understand the mechanisms underlying the activation of follistatin by activin A, follistatin transcription was evaluated in gonadotrope-derived αT3-1 cells. Transient transfection experiments established that follistatin-luciferase plasmids that incorporate up to 2.86 kb of the upstream region of the rat follistatin gene are not induced by activin A in αT3-1 cells. On the other hand, plasmids that incorporate intron 1 are responsive to activin A and induced by a constitutively active form of ALK4. These experiments ultimately identified a conserved Smad-binding element (SBE1) in intron 1, between +1791 and +1795. In αT3-1 cells treated with activin A, SBE1 preferentially recruits Smad3, but not Smad2, and mediates Smad3-dependent activation of follistatin transcription. shRNA knockdown of endogenous Smad3 in these cells compromises SBE1-mediated transcription in response to activin A and interferes with its ability to positively regulate follistatin mRNA levels. The findings of the current work illustrate the critical role of intron 1 of the follistatin gene in mediating Smad-dependent effects of activin and regulating the expression level of this gene in some cell types, such as pituitary cells of gonadotrope lineage. Follistatins exert critical autocrine or paracrine control in many tissues by binding and bio-neutralizing activin and several other transforming growth factor-β ligands. In the pituitary, activin acts locally to induce follistatin expression and thus modulate its own actions. This local feedback loop safeguards against excessive activin signaling and maintains the necessary balance of activin and follistatin tone. To better understand the mechanisms underlying the activation of follistatin by activin A, follistatin transcription was evaluated in gonadotrope-derived αT3-1 cells. Transient transfection experiments established that follistatin-luciferase plasmids that incorporate up to 2.86 kb of the upstream region of the rat follistatin gene are not induced by activin A in αT3-1 cells. On the other hand, plasmids that incorporate intron 1 are responsive to activin A and induced by a constitutively active form of ALK4. These experiments ultimately identified a conserved Smad-binding element (SBE1) in intron 1, between +1791 and +1795. In αT3-1 cells treated with activin A, SBE1 preferentially recruits Smad3, but not Smad2, and mediates Smad3-dependent activation of follistatin transcription. shRNA knockdown of endogenous Smad3 in these cells compromises SBE1-mediated transcription in response to activin A and interferes with its ability to positively regulate follistatin mRNA levels. The findings of the current work illustrate the critical role of intron 1 of the follistatin gene in mediating Smad-dependent effects of activin and regulating the expression level of this gene in some cell types, such as pituitary cells of gonadotrope lineage. Activins are members of the evolutionarily conserved transforming growth factor-β (TGF-β) 3The abbreviations used are:TGFtransforming growth factorFBSfetal bovine serumntnucleotidesDTTdithiothreitolChIPchromatin immunoprecipitation assayMOPS4-morpholinepropanesulfonic acidCMVcytomegalovirusGFPgreen fluorescent proteinARFactivin-responsive fragmentSBESmad-binding elementRAPrat anterior pituitary cellsGAPDHglyceraldehyde-3-phosphate dehydrogenasemAbmonoclonal antibodyshRNAshort hairpin RNA.3The abbreviations used are:TGFtransforming growth factorFBSfetal bovine serumntnucleotidesDTTdithiothreitolChIPchromatin immunoprecipitation assayMOPS4-morpholinepropanesulfonic acidCMVcytomegalovirusGFPgreen fluorescent proteinARFactivin-responsive fragmentSBESmad-binding elementRAPrat anterior pituitary cellsGAPDHglyceraldehyde-3-phosphate dehydrogenasemAbmonoclonal antibodyshRNAshort hairpin RNA. superfamily of factors implicated in the control of a wide array of cellular processes of embryonic and adult tissues (1Vale W. 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The present study was undertaken to further evaluate the mechanism involved in the transcriptional activation of the follistatin gene by activin and to identify activin-responsive regulatory elements. The mouse αT3-1 gonadotrope cell line, which expresses endogenous follistatin, was used as a cellular model. The results indicate that activin induction of the follistatin gene is mediated by a conserved Smad-binding element that localizes to the first intron. Primary Cells and Cell Lines—Primary rat anterior pituitary (RAP) cells were prepared by collagenase-mediated dispersion of anterior pituitaries obtained from male Sprague-Dawley rats (180-200 g) as previously described (24Bilezikjian L.M. Corrigan A.Z. Vaughan J.M. Vale W.W. Endocrinology. 1993; 133: 2554-2560Crossref PubMed Scopus (71) Google Scholar). The dispersed RAP cells were seeded on tissue culture plates and maintained at 7.5% CO2 in a humidified 37 °C incubator in a specially formulated medium (designated βPJ) containing appropriate growth factors and 2% fetal bovine serum (FBS) (24Bilezikjian L.M. Corrigan A.Z. Vaughan J.M. Vale W.W. Endocrinology. 1993; 133: 2554-2560Crossref PubMed Scopus (71) Google Scholar). The cells were allowed to recover for at least 3 days before initiating experiments. The mouse gonadotrope-derived αT3-1 (42Horn F. Bilezikjian L.M. Perrin M.H. Bosma M.M. Windle J.J. Hille B. Vale W.W. Mellon P.L. Mol. Endocrinol. 1990; 5: 347-355Crossref Scopus (145) Google Scholar) and the human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 2 mm glutamine. Analysis of Follistatin Transcript Levels by RNase Protection Assays and Real-time PCR—On the 4th day following dispersion, RAP cells (107 per 10-cm tissue culture dish) were washed and equilibrated overnight in with 0.2% FBS in βPJ medium, then washed again and treated in fresh medium with activin A or vehicle. Total RNA from either the nuclear or cytoplasmic compartment of 107 RAP cells was isolated by lysis in ice-cold Nonidet P-40 buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40). The nuclei were collected by a 10-min centrifugation at 1000 × g, then subjected to sequential digestion with RNase-free DNase (Promega, Madison, WI) for 10 min at 37 °C and proteinase K (EM Science, Gibbstown, NJ) for 30 min at 42 °C. The cytoplasmic fractions were treated for 1 h at 42 °C with 100 μg/ml proteinase K. The RNA recovered from each compartment was used in its entirety for each hybridization reaction to measure follistatin transcripts. In the case of αT3-1 cells, total RNA was extracted with the RNeasy kit (Qiagen, Hilden, Germany), and ∼50 μg was used to evaluate follistatin transcript levels. The rat and mouse follistatin cDNA templates were constructed by subcloning into pBluescript IISK (Stratagene, La Jolla, CA) the corresponding genomic fragments spanning the junction of exon 3 and intron 3. Antisense riboprobes corresponding to rat or mouse follistatin were synthesized in the presence of [α-32P]UTP (3000 Ci/mmol) using T3 or T7 RNA polymerase from templates linearized with either HindIII or BamHI, respectively. The rat follistatin riboprobe protects 463 or 203 nt corresponding to primary (unprocessed) or mature mRNA transcript, respectively. The mouse follistatin riboprobe protects 470 nt of primary transcript or 190 nt of mature mRNA. Rat and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense riboprobes were synthesized using T3 RNA polymerase to yield a protected fragment of 136 nt, as previously described (28Bilezikjian L.M. Corrigan A.Z. Blount A.L. Vale W.W. Endocrinology. 1996; 137: 4277-4284Crossref PubMed Scopus (75) Google Scholar). The samples were resolved on 5% polyacrylamide, 8 m urea gels, and band intensity was quantified using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the Image-Quant 4.0 software package. Follistatin transcript levels were normalized to internal GAPDH levels and are reported as means + S.E. Plasmid Constructs—Expression plasmids encoding N-terminally Myc-tagged human (h) Smad2, -3, or -4 and the constitutively active ALK4 (caALK4) were generated by PCR and subcloned into the pCS2+ expression vector. The numeric designations of the rat genomic fragments used to describe the follistatin-luciferase plasmids are relative to the most downstream transcription initiation site observed in αT3-1 cells 4L. M. Bilezikjian, unpublished results. and previously designated as "α" (35Miyanaga K. Shimasaki S. Mol. Cell. Endocrinol. 1993; 92: 99-109Crossref PubMed Scopus (41) Google Scholar). The rFS(2.9)-luc reporter was constructed by subcloning the KpnI/EcoNI (-2864/+136) fragment of the rat follistatin gene into the pGL2 basic luciferase vector (Promega). The rFS(2.9i)-luc plasmid contains the entire first intron (+227/+2097) of the rat follistatin gene just downstream of the -2864/+136 fragment. To exclude the ATG initiation site from this construct, a 90-bp fragment overlapping exon 1 was deleted by replacing the Aat II/Blp I (-3/+758) fragment with two PCR-amplified fragments corresponding to -3/+136 (forward and reverse primers: 5′-TCTAGATTTAAAGC and 5′-ACTAGTGGCAGGCGCGGGGCGAGGA) and +227/+758 (forward and reverse primers: 5′-ACTAGTGAGTGGAGGGGATGCGCCCA and 5′-ACTTCGGGCTAATAATTTGGTTTG) that were ligated via an engineered SpeI site. The rFS(0.3i)-luc and rFS(0.3iP)-luc plasmids were generated by digesting rFS(2.9i)-luc at the unique Mlu I site to remove the portion upstream of -312 or at the PmlI site to delete 313 bp from the 3′-end of intron 1. The rFS(0.3i)-luc plasmid was further modified using the Erase-A-Base System (Promega) to serially truncate the 3′-end of intron 1 and generate rFS(0.3i45)-luc, rFS(0.3i91)-luc and rFS(0.3i115)-luc (described in Fig. 4). The rFS(0.3i45)-luc plasmid was further digested with SpeI and Pml I to remove most of intron 1 (+228/+1784) and generate rFS(0.3ex45)-luc. Point mutations within the SBE1 site of the rFS(0.3ex45)-luc plasmid were introduced using a PCR approach with upstream primers containing the indicated substitutions (described in Fig. 4) and a reverse primer within the pGL2 vector. All constructs were subjected to sequence analysis. Transfections and Luciferase Reporter Assays—The αT3-1 cells were seeded in poly-l-lysine-coated 12-well tissue culture plates at a density of 3 × 105 cells/well in 2 ml of complete medium (Dulbecco's modified Eagle's medium, 10% FBS, and 2 mm glutamine). The cells were transfected the next morning by incubating them for 6 h with a mix of the Superfect Transfection Reagent (Qiagen; Hilden, Germany), 0.6 μg/well of luciferase reporter plasmid and 0.2 μg/well cytomegalovirus (CMV)-β-galactosidase (β-Gal) plasmid as an internal control. Where indicated, varying amounts of expression plasmids encoding Myc-tagged Smads, caALK4, or empty vector were co-transfected along with the reporters. At the end of the 6-h transfection period, the cells were washed and treated with vehicle or activin A in Dulbecco's modified Eagle's medium supplemented with 2% FBS and 2 mm glutamine. The cells were harvested 15 h later in lysis buffer (1% Triton X-100, 25 mm glycylglycine, pH 7.8, 15 mm MgSO4, 4 mm EGTA, and 1 mm DTT). Luciferase reporter activity was measured using d-luciferin luciferase substrate (Biosynth, Naperville, IL) with a Lumimark microplate luminometer (Bio-Rad) or a Lumat LB 9507 (EG&G Berthold, Bad Wildbad, Germany) and normalized to that of CMV-β-Gal. Reported data correspond to luciferase/β-Gal ratios of each plasmid relative to the activity of the pGL2 basic vector. Chromatin Immunoprecipitation (ChIP)—The method used for ChIP analysis was essentially as described previously (43Asahara H. Santoso B. Guzman E. Du K. Cole P.A. Davidson I. Montminy M. Mol. Cell. Biol. 2001; 21: 7892-7900Crossref PubMed Scopus (84) Google Scholar). Activin A or vehicle-treated αT3-1 cells were cross-linked with 1% formaldehyde for 15 min at room temperature. The cells were lysed by incubating them for 10-15 min on ice in lysis buffer (25 mm HEPES, pH 7.8, 1.5 mm MgCl2, 10 mm KCl, 0.1% Nonidet P40, 1 mm DTT, and protease inhibitors). The nuclear fraction that was recovered by centrifugation (5 min at 5000 × g) was resuspended in ChIP buffer (50 mm HEPES, pH 7.8, 140 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitors) and sonicated on ice (Misonix XL200 ultrasonic cell disruptor) to achieve an average chromatin length of 500-1000 bp. The sonicated samples were precleared by incubation with protein A-Sepharose in the presence of 8 μg/ml salmon sperm DNA, 0.3% normal rabbit serum, and 0.05% bovine serum albumin followed by centrifugation. The material recovered from the equivalent of ∼107 αT3-1 cells was incubated overnight at 4 °C with 5 μl of either normal rabbit IgG, anti-hSmad2/3, or anti-hSmad2/3 preabsorbed with the peptide antigen along with protein A-Sepharose. The protein A-purified rabbit anti-hSmad2/3 used for these experiments is directed against a peptide within the linker of hSmad2 (amino acids 199-215), which is conserved in hSmad3. The protein A-Sepharose beads were washed sequentially once with ChIP buffer, twice with ChIP buffer containing 0.5 m NaCl, once with 0.25 m LiCl buffer (20 mm Tris-HCl, pH 8.0, 0.5% Nonidet P-40, 0.5% deoxycholate, 1 mm EDTA), and finally, twice with TE buffer (10 mm Tris-HCl, pH 8, 1 mm EDTA). The specifically bound complexes were eluted from the protein A-Sepharose beads by two 15-min incubations at 65 °C with TE elution buffer (10 mm Tris-HCl, pH 8, 1 mm EDTA, 1% SDS). The immunoprecipitated complexes and the starting material (input) were incubated overnight at 65 °C to reverse cross-linking, then treated with proteinase K and purified using QIAquick Spin Columns (Qiagen, Hilden, Germany). The DNA samples were recovered in 50 μl of 10 mm Tris-HCl, pH 8.5, and analyzed by semiquantitative PCR using primers that amplify a fragment of 185 bp overlapping the SBE1 site within intron 1 of the endogenous mouse follistatin gene (forward: 5′-GTCGCTGCAGGTTATGAAATGG and reverse: 5′-AAAGGGGAGAGTGGGGAAGGAC). The amplified fragment was then resolved on a 2% agarose gel and analyzed by ethidium bromide staining. Alternatively, DNA from 1-2 μl of each sample was quantified by real-time PCR using the SYBR GREEN PCR Master Mix and the ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, CA). The fragment containing the SBE1 site was amplified using primers flanking the site (forward: 5′-AACAGTCTAGTAAAAGTCAATGCAAGCT and reverse: 5′-TGCGCCCCAGCCATAT). A primer set that amplifies a fragment ∼5-kb upstream of the transcription start site of the mouse follistatin gene (forward: 5′-AGATAGAGATCCCACCACAGAACAA and reverse: 5′-GGATGGACTTGGGTGGTATCTGTA) or a fragment of the mouse β-actin gene (forward: 5′-TTCCCTTCCACAGGGTGTGA and reverse: 5′-ACATAGGAGTCCTTCTGACCCATT) were used as internal controls. Primer pairs flanking the upstream putative Smad3 site at -1604 (forward: 5′-CGGCTGTATTTCGGGATCTATT and reverse: 5′-ACTGCAGGAGATAGTGCTAATCTTTTAAT) and Smad4 site at -895 (forward: 5′-GAAAGGGAGAGGGCGAGACT and reverse: 5′-CCCTCGGGCTCCACAAGT) were used to amplify the corresponding fragments. Oligonucleotide Precipitation Assays—For these experiments, a lentiviral delivery system was used to facilitate the expression of Myc-tagged Smads in αT3-1. The N-terminally Myc-tagged hSmad2, -3, or -4 cDNAs were subcloned upstream of an IRES GFP marker in the pCSC-SP-PW-IRES/GFP lentiviral transfer vector (generously provided by Dr. Inder Verma, Salk Institute, La Jolla, CA). The GFP-expressing pCSC-SP-PW-IRES/GFP empty vector was used as control. Recombinant lentiviruses were produced by co-transfecting HEK293T cells with the Smad expression or control transfer plasmid and three additional plasmids required for packaging (pMDL, pRev, and pVSVG) using polyethylenimine as the transfection reagent, as described (44Tiscornia G. Singer O. Verma I.M. Nat. Protoc. 2006; 1: 241-245Crossref PubMed Scopus (715) Google Scholar). The supernatants containing the viral particles were collected 48 h after transfection, filtered through a 0.45-μm filter, and concentrated by ultracentrifugation for 2.5 h at 50,000 × g. Relative titers were assessed by monitoring the percentage of GFP-positive HEK293T cells infected with serial dilutions of the viral preparations. For oligonucleotide precipitation experiments, αT3-1 cells (5 × 106/10 cm dish) were infected in the presence of 8 μg/ml polybrene (Sigma) with lentiviral vectors to express only GFP as a control or combinations of Myc-Smad2 and -4 or Myc-Smad3 and -4. The amount of virus necessary to achieve >90% GFP-positive αT3-1 cells was predetermined by performing serial dilutions of equivalent titers of the viral preparations. Protein expression was allowed to progress for 5 days at which time the cells were replated into four 10-cm dishes and allowed to grow for two more days. The cells were supplemented with fresh medium and treated for 30 min with 1 nm activin A or vehicle in duplicate. Lysates of αT3-1 cells were prepared by brief sonication in lysis buffer (25 mm Tris-HCl, pH 7.5, 0.1% Triton X-100, 10% glycerol, 1 mm MgCl2, 0.5 mm EDTA, 100 mm NaCl, 5 mm NaF, 1 mm Na4P2O7, 1 mm DTT, and protease inhibitors) followed by a 10-min centrifugation at 12,000 × g at 4 °C. The supernatant obtained from each 10-cm dish (850 μg) was incubated for 2 h at 4 °C with 1 μg of biotinylated double-stranded oligonucleotides, precoupled to streptavidin-agarose beads (Pierce), in the presence of 8 μg of poly(dI-dC) (Sigma). The agarose beads were washed three times by centrifugation, and specifically bound proteins were recovered and subjected to Western analysis. The samples were resolved under reducing conditions using 10% NuPAGE SDS gels (Invitrogen) and MOPS as the running buffer then transferred to nitrocellulose membranes. After blocking the membranes with 5% BLOTTO (Pierce), Myc-Smads were detected using an anti-Myc monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ). Immune complexes were then visualized with SuperSignal West Pico chemiluminescence substrate (Pierce). The experiments were performed using biotinylated wild type (forward: 5′-CAAGCTGCACGTGTTGTGTCTGGGTCACTGGTAACTGACATTGATATGGCTAGGCGCAGCGGCTGCTGCTC; reverse: 5′-biotin-GAGCAGCAGCCGCTGCGCCTAGCCATATCAATGTCAGTTACCAGTGACCCAGACACAACACGTGCAGCTTG) and SBE1 mutant (forward 5′-CAAGCTGCACGTGTTGTaatTGGGTCACTGGTA ACTGACATTGATATGGCTAGGCGCAGCGGCTGCTGCTC; antisense 5′-biotin-GAGCAGCAGCCGCTGCGCCTAGCCATATCAATGTCAGTTACCAGTG
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