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• W2039717963 abstract "Laminin is a major component of the extracellular matrix. The laminin γ1 chain is the least variant component of the laminin heterotrimeric assembly. The laminin γ1 chain gene (LAMC1) expression is induced by several factors, including transforming growth factor-β (TGF-β). LAMC1 promoter contains a highly conserved transcriptional element, bcn-1. We screened cDNA libraries with the yeast one-hybrid system to identify transcriptional factors that are recognized by the bcn-1 motif. Using this strategy we isolated the basic helix-loop-helix/leucine zipper (bHLHzip) E-box-binding transcription factor, TFE3. Until now, the E-box was the only element known to recruit the bHLHzip transcription factors. Although the bcn-1 element only remotely resembles the E-box sequence, we show that TFE3 binds and activates the bcn-1 element. TFE3 cooperates with Smad proteins in the activation of the LAMC1 promoter in cells, an effect that is critically dependent not only on the bcn-1 element but also on one of the Smad-binding elements (SBE). The cooperative induction of the LAMC1 promoter and the endogenous LAMC1 gene by TFE3 and Smad3 is augmented by the TGF-β signaling pathway. Thus, the bcn-1 is a novel TFE3-dependent TGF-β target element that regulates LAMC1 gene expression. Laminin is a major component of the extracellular matrix. The laminin γ1 chain is the least variant component of the laminin heterotrimeric assembly. The laminin γ1 chain gene (LAMC1) expression is induced by several factors, including transforming growth factor-β (TGF-β). LAMC1 promoter contains a highly conserved transcriptional element, bcn-1. We screened cDNA libraries with the yeast one-hybrid system to identify transcriptional factors that are recognized by the bcn-1 motif. Using this strategy we isolated the basic helix-loop-helix/leucine zipper (bHLHzip) E-box-binding transcription factor, TFE3. Until now, the E-box was the only element known to recruit the bHLHzip transcription factors. Although the bcn-1 element only remotely resembles the E-box sequence, we show that TFE3 binds and activates the bcn-1 element. TFE3 cooperates with Smad proteins in the activation of the LAMC1 promoter in cells, an effect that is critically dependent not only on the bcn-1 element but also on one of the Smad-binding elements (SBE). The cooperative induction of the LAMC1 promoter and the endogenous LAMC1 gene by TFE3 and Smad3 is augmented by the TGF-β signaling pathway. Thus, the bcn-1 is a novel TFE3-dependent TGF-β target element that regulates LAMC1 gene expression. Laminin is a large glycoprotein component of the extracellular matrix localized in basement membranes (1.Deutzmann R. Schlondorff D. Bonventre J.V. Molecular Nephrology: Kidney Function in Health and Disease. Dekker, New York1995: 41-59Google Scholar). Laminin is a multifunctional protein that regulates cell-to-matrix interactions, including cell adhesion, spreading, migration, and differentiation. Laminin exerts its effects on cellular behavior by interacting with cellular receptors that recognize specific sites on the laminin molecule. Laminin exhibits all the attributes of an extracellular signaling molecule that regulates cellular architecture, for example that of glomeruli (2.Abrahamson D.R. John P.L.S. Kidney Int. 1993; 43: 73-78Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 3.Hansen K. Abrass C.K. Pathobiology. 1999; 67: 84-91Crossref PubMed Scopus (17) Google Scholar). The best studied laminin receptors are the members of the integrin family of proteins (1.Deutzmann R. Schlondorff D. Bonventre J.V. Molecular Nephrology: Kidney Function in Health and Disease. Dekker, New York1995: 41-59Google Scholar). Laminin is composed of three polypeptide chains, α, β, and γ, held together by disulfide bonds, forming a cruciform structure (4.Tunggal P. Smyth N. Paulsson M. Ott M.C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar). There are five α (α1–5), three β (β1–3), and three γ (γ1–3) chains that account for the twelve known laminin trimeric assemblies (Laminin 1–12) (4.Tunggal P. Smyth N. Paulsson M. Ott M.C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar). The γ1 chain, found in ten of the twelve known trimeric laminin isoforms, is the most widely expressed laminin chain (4.Tunggal P. Smyth N. Paulsson M. Ott M.C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar). Laminin γ1 is required for basement membrane formation, and its absence causes early lethality in mouse embryos (5.Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. J. Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (427) Google Scholar, 6.Murray P. Edgar D. J. Cell Biol. 2000; 150: 1215-1221Crossref PubMed Scopus (116) Google Scholar). These observations suggest that the synthesis of γ1 chain is essential for the laminin heterotrimeric assembly. LAMC1 gene expression is responsive to extracellular signals such as TGF-β 1The abbreviations used are: TGF-βtransforming growth factor-β3-AT3-aminotrizoleSBESmad-binding elementMOPS4-morpholinepropanesulfonic acidTFE3transcription factor E3bHLHzipbasic helix-loop-helix/leucine zipperPAI-1plasminogen activator inhibitor-1TβRI and -IIserine/threonine kinase receptors I and IIR-Smadreceptor-activated SmadLAMC1laminin γ1 chain (7.Suzuki H. O'Neill B.C. Suzuki Y. Denisenko O.N. Bomsztyk K. J. Biol. Chem. 1996; 271: 18981-18988Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), interleukin-1β (8.Richardson C.A. Gordon K.L. Couser W.G. Bomsztyk K. Am. J. Physiol. 1995; 268: F273-F278PubMed Google Scholar), and glucose (9.Phillips S.L. DeRubertis F.R. Craven P.A. Diabetes. 1999; 48: 2083-2089Crossref PubMed Scopus (17) Google Scholar). Because of its vital role, transcription of the laminin γ1 chain gene has been intensely studied (7.Suzuki H. O'Neill B.C. Suzuki Y. Denisenko O.N. Bomsztyk K. J. Biol. Chem. 1996; 271: 18981-18988Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 10.Kedar V. Freeze E. Hempel F. Indian J. Exp. Biol. 1996; 34: 939-944PubMed Google Scholar, 11.Kedar V. Freese E. Hempel F.G. Mol. Brain Res. 1997; 47: 87-98Crossref PubMed Scopus (9) Google Scholar, 12.Ogawa K. Burbelo P.D. Sasaki M. Yamada Y. J. Biol. Chem. 1988; 263: 8384-8389Abstract Full Text PDF PubMed Google Scholar, 13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar, 14.Suzuki H. Denisenko O.N. Suzuki Y. Schullery D.S. Bomsztyk K. Am. J. Physiol. 1998; 275: F518-F526PubMed Google Scholar). The 5′-flanking regions of the human (15.Kallunki T. Ikonen J. Chow L.T. Kallunki P. Tryggvanson K. J. Biol. Chem. 1991; 266: 221-228Abstract Full Text PDF PubMed Google Scholar), murine (12.Ogawa K. Burbelo P.D. Sasaki M. Yamada Y. J. Biol. Chem. 1988; 263: 8384-8389Abstract Full Text PDF PubMed Google Scholar), and rat (13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar) laminin γ1 chain gene have been cloned. Although the human and the rodent promoters share only a small degree of sequence similarity, they have several common features that may be functionally be similar. (i) There are no TATA or CAAT boxes in either promoter, motifs common in genes transcribed by RNA polymerase II (16.Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2210) Google Scholar). Although uncommon, TATA-less promoters have been described for many genes (17.Smale S.T. Baltimore D. Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1151) Google Scholar). Similar to other TATA-less promoters (18.Parks C.L. Shenk T. J. Biol. Chem. 1996; 271: 4417-4430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), the rat, mouse, and human LAMC1 promoters have multiple transcription initiation sites (13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar) providing greater flexibility to initiate transcription. (ii) The murine and rat LAMC1 promoters have a number of repeats of a unique consensus sequence, CCCG(T)CCCA(T)CCT. An identical motif is present in the human laminin γ1 chain gene promoter as well (12.Ogawa K. Burbelo P.D. Sasaki M. Yamada Y. J. Biol. Chem. 1988; 263: 8384-8389Abstract Full Text PDF PubMed Google Scholar, 13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar,15.Kallunki T. Ikonen J. Chow L.T. Kallunki P. Tryggvanson K. J. Biol. Chem. 1991; 266: 221-228Abstract Full Text PDF PubMed Google Scholar). (iii) Rat, mouse, and human promoters contain a number of “GC”-rich motifs similar to the binding site (GGGCGG) for the transcriptional factor, Sp1 (19.Briggs M.R. Kadonaga J.T. Bell S.P. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1059) Google Scholar, 20.Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1255) Google Scholar). (iv) Finally, the rat, murine, and human promoters contain an identical bcn-1 transcriptional element (CCCCGCCCACCTCGCGC) (7.Suzuki H. O'Neill B.C. Suzuki Y. Denisenko O.N. Bomsztyk K. J. Biol. Chem. 1996; 271: 18981-18988Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). transforming growth factor-β 3-aminotrizole Smad-binding element 4-morpholinepropanesulfonic acid transcription factor E3 basic helix-loop-helix/leucine zipper plasminogen activator inhibitor-1 serine/threonine kinase receptors I and II receptor-activated Smad laminin γ1 chain Because the bcn-1 element may play an important role in the regulation of LAMC1 gene expression, in the present study we set out to identify and characterize transcription factors that act from this element. Using the yeast one-hybrid screen we identified TFE3 as one of the factors that regulates the bcn-1 element. We show that TFE3 cooperates with Smad proteins in the activation of the LAMC1gene, an effect that is bcn-1- and SBE-dependent and is augmented by the TGF-β signaling pathway. Rat mesangial cells, SI−MC cells, were cultured and maintained as described previously (21.Abrass C.K. Spicer D. Raugi G.J. Kidney Int. 1995; 47: 25-37Abstract Full Text PDF PubMed Scopus (54) Google Scholar). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 200 μg/ml l-glutamine. Cells were maintained at 37 °C in 5% CO2 in air and were passed every 5–7 days by trypsin. Yeast shuttle vectors, pHISi and pLacZi, were purchased from CLONTECH (Palo Alto, CA). 3-Aminotrizole (3-AT) was purchased from Sigma Chemical Co. (St. Louis, MO), and SuperFect was obtained from Qiagen (Valencia, CA). The rat mesangial cells used for construction of the library were established from collagenase-treated glomeruli, and cells were characterized (8.Richardson C.A. Gordon K.L. Couser W.G. Bomsztyk K. Am. J. Physiol. 1995; 268: F273-F278PubMed Google Scholar, 22.Adler S. Baker P.J. Johnson R.J. Ochi R.F. Pritzl P. Couser W.G. J. Clin. Inv. 1986; 77: 762-770Crossref PubMed Scopus (190) Google Scholar) and maintained as described previously (14.Suzuki H. Denisenko O.N. Suzuki Y. Schullery D.S. Bomsztyk K. Am. J. Physiol. 1998; 275: F518-F526PubMed Google Scholar). Total cellular RNA was prepared from phorbol 12-myristate 13-acetate-treated (1 h) rat mesangial cells as described previously (23.Okayama H. Kawaichi M. Brownstein M. Lee F. Yokota T. Arai K. Methods Enzymol. 1987; 154: 3-28Crossref PubMed Scopus (286) Google Scholar). Poly(A)+RNA was reverse-transcribed by using an oligo(dT) primer with an XhoI site. The RNA strand of the mRNA-cDNA hybrid was replaced with the corresponding DNA strand by using Escherichia coli RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (23.Okayama H. Kawaichi M. Brownstein M. Lee F. Yokota T. Arai K. Methods Enzymol. 1987; 154: 3-28Crossref PubMed Scopus (286) Google Scholar), and the cDNA was ligated to EcoRI linkers after both termini were blunt-ended. After cleavage with EcoRI and XhoI, the cDNA was ligated to EcoRI/SalI-digested yeast expression plasmid pGAD424 (CLONTECH, Palo Alto, CA). The ligation products were purified by ethanol precipitation and electro-transformed into E. coli DH10B (Invitrogen, Rockville, MD) with E. coli Pulser (Bio-Rad, Hercules, CA) to generate the pGAD-MC cDNA library. The yeast one-hybrid screen analysis was carried out according to the manufacturer's protocol (MATCHMAKER One-Hybrid System, CLONTECH). Briefly, three tandem copies of the 17-bp bcn-1 motif (5′-ccccgcccacctcgcgc-3′) were inserted upstream of the His3 and LacZ reporter genes by designing sense and antisense 3× bcn-1 oligonucleotides with EcoRI and XbaI or SalI sites at the ends. Sense and antisense oligonucleotides were annealed and subcloned into the EcoRI/XbaI-digested, pHISi-1 or EcoRI/SalI-digested pLacZi reporter plasmids (CLONTECH). Inserts were sequenced to confirm the presence and integrity of the bcn-1 elements. Both bcn-1-reporter constructs were integrated into the genome of YM4271 strain to generate bcn-1-reporter strains. Background HIS3 expression was ablated using 10 mm 3-aminotrizole (3-AT) (Sigma). Screening bcn-1-reporter strains with a pGAD-MC cDNA library identified genes encoding bcn-1-binding proteins. Plasmids were isolated from yeast clones capable of growing in the presence of 10 mm 3-AT in (−) leucine and (−) histidine media. Plasmids from these clones were transformed into competent DH5α bacteria, and each cDNA insert was sequenced and compared with known sequences in the GenBankTM data base. False positives were excluded using the LacZ reporter gene assay. The partial or complete cDNAs of TFE3 were used as a template for in vitro transcription by SP6 or T7 DNA-dependent RNA polymerases to generate mRNAs for in vitro translation as previously described (24.Gurevich V.V. Pokrovskaya I.D. Obukhova T.A. Zozulya S.A. Anal. Biochem. 1991; 195: 207-213Crossref PubMed Scopus (132) Google Scholar). In vitro translation in a rabbit reticulocyte cell-free system was performed according to the manufacturer's protocol (Promega, Madison, WI). To confirm that the bcn-1 element binds to TFE3, 3 μl of cell-free system containing 35S-labeled proteins was incubated in 150 μl of binding buffer for 30 min (4 °C) with 0.5 μg of wild-type bcn-1 dimer double-stranded oligonucleotide with biotin on the 5′-end of the sense strand. After the binding reaction, the DNA·protein complexes were pulled-down with streptavidin-agarose beads, and the beads were washed three times with 1 ml of washing buffer (10 mm Tris HCl, pH 7.5, 100 mm KCl, 2 mm MgCl2 and 0.1%Triton X-100). 35S-Labeled proteins eluted with SDS-loading buffer (60 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol) and were analyzed by SDS-PAGE and autoradiography. TFE3L and TFE3S were subcloned into pEX vector, a derivative of pEGFP-N1 (CLONTECH), after cutting it out from pSV2A-TFE3 (gift from Dr. Kathryn Calame) (25.Roman C. Cohn L. Calame K. Science. 1991; 254: 94-97Crossref PubMed Scopus (99) Google Scholar) with NheI and BamHI. pEXL-Smad3, pEXL-Smad4, and pCMV-TβRI-T204D were described previously (gift from Dr. Xianxin Hua) (26.Hua X. Miller Z.A. Wu G. Shi Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13130-13135Crossref PubMed Scopus (134) Google Scholar). The LAMC1 promoter fragment spanning the −1077 to −20 region of the gene (relative to first ATG codon), designated −1077/−20 LAMC1, was constructed by PCR amplification of the LAMC1−1104/+35 5′-flanking promoter region (13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar). The deletion constructs of LAMC1 promoter, −506/−20 LAMC1, −293/−20 LAMC1, −1077/−529 LAMC1, were generated by PCR using appropriate primers and the −1077/−20 LAMC1 promoter as a template. The PCR products were ligated into pGEM-T Easy vector (Promega) and digested with SacI and BglII. The SacI-BglII fragments were ligated into pGL3 basic vector cut with SacI-BglII using a Rapid DNA Ligation Kit (Roche Molecular Biochemicals, Indianapolis, IN). −239/−20 LAMC1 was constructed by digesting the −1077/−20LAMC1 promoter with XhoI and BglII, and the fragments were subcloned into XhoI- and BglII-digested pGL3 basic vector. All constructs were confirmed by DNA sequencing. Site-directed mutagenesis was done using the QuikChange site- directed mutagenesis kit (Stratagene, La Jolla, CA). The rat LAMC1 promoter (−1077 to −20, relative to the first codon) was used as a template. Mutagenesis of the two bcn-1 bases (see boldface letters below) required for protein binding was performed using the following oligonucleotides: sense, 5′-CTCCACCGCCCCGCCCGGCTCGCGCCCTTCCC-3′; and antisense, 5′-GGGAAGGGCGCGAGCCGGGCGGGGCGGTGGAG-3′. The site-directed mutagenesis of the four SBE bases, (see boldface below) was performed using the following oligonucleotides: sense, 5′-GGGGAGGGCGAGGTCACGCCTTGCACTGCGTGGTTGCGG-3′; and antisense, 5′-CCGCAACCACGCAGTGCAAGGCGTGACCTCGCCCTCCCC-3′ for SBE1, with sense, 5′-CCTCATGAAGTCAAACTTGAGATTCCGAGCCACCTCACCG-3′; and antisense, 5′-CGGTGAGGTGGCTCGGAATCTCAAGTTTGACTTCATGAGG-3′ for SBE2. All mutations were verified by DNA sequencing. SI−MC cells were plated at 4 × 105 cells/well in 6-well plates and incubated in RPMI with 10% fetal bovine serum for 24 h. Cells were transiently transfected with 2.0 μg of reporter plasmid, effector expression plasmid (indicated in each experiment), and 0.1 μg of pRL-null (Promega) using SuperFect transfection reagent (Qiagen). Cell extracts were prepared 24 h later by passive lysis buffer (Promega), and luciferase activity was assayed by Dual Reporter Assay System (Promega) (27.Shnyreva M. Schullery D.S. Suzuki H. Higaki Y. Bomsztyk K. J. Biol. Chem. 2000; 275: 15498-15503Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). SI−MC cells were plated at 1 × 106 cells/plate in 100-mm plates and grown in RPMI media with 10% fetal bovine serum for 24 h. Cells were transiently transfected with 10.0 μg of expression vector containing TFE3L, Smad3, TβRI-T204D, and complementary empty vector using SuperFect transfection reagent (Qiagen). After 24 h, total RNA was extracted as described before (28.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with some modification. Briefly, after lysing cells with 6 m urea/4 m LiCl, total RNA was extracted with phenol/chloroform and 100% ethanol. After denaturing, 10 μg of total RNA was electrophoresed on 1% agarose gel with 2.2m formaldehyde and 20 mm MOPS, pH 7.0. After electrophoresis, RNA was transferred overnight onto a nylon membrane (Nytran-N nylon membrane, Schleicher & Schuell, Dassel, Germany) in 10× standard saline citrate (1× SSC: 150 mm NaCl, 15 mm sodium citrate) by a rapid downward transfer system (Turboblotter, Schleicher & Schuell). After cross-linking of RNA, hybridization was done with 32P-labeled LAMC1cDNA probe (14.Suzuki H. Denisenko O.N. Suzuki Y. Schullery D.S. Bomsztyk K. Am. J. Physiol. 1998; 275: F518-F526PubMed Google Scholar). Membranes were washed, and signals were measured using a phosphorimaging device (Cyclone, Packard Instrument Co., Meriden, CT). The rodent and human laminin γ1 chain gene (LAMC1) promoter contains a transcriptional element, bcn-1, that binds nuclear proteins from mesangial cells (7.Suzuki H. O'Neill B.C. Suzuki Y. Denisenko O.N. Bomsztyk K. J. Biol. Chem. 1996; 271: 18981-18988Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 13.O'Neill B.O. Suzuki H. Loomis W.P. Denisenko O. Bomsztyk K. Am. J. Physiol. 1997; 273: F411-F420PubMed Google Scholar, 14.Suzuki H. Denisenko O.N. Suzuki Y. Schullery D.S. Bomsztyk K. Am. J. Physiol. 1998; 275: F518-F526PubMed Google Scholar). We generated a cDNA fusion library from rat mesangial cells for a screen in yeast one-hybrid system to identify factors that bind the bcn-1 element. One of the cDNA clones isolated in this screen encoded the transcription factor E3 (TFE3) (29.Beckmann H. Su L.K. Kadesch T. Genes Dev. 1990; 4: 167-179Crossref PubMed Scopus (354) Google Scholar). TFE3 is composed of a basic helix-loop-helix region and a leucine zipper (bHLHzip). Both the basic helix-loop-helix region and the leucine zipper domains are essential for DNA binding and dimerization (25.Roman C. Cohn L. Calame K. Science. 1991; 254: 94-97Crossref PubMed Scopus (99) Google Scholar, 29.Beckmann H. Su L.K. Kadesch T. Genes Dev. 1990; 4: 167-179Crossref PubMed Scopus (354) Google Scholar). The members of the bHLHzip family of transcription factors, including TFE3, bind directly the E-box DNA motif. Isolation of the TFE3 was unexpected, because the bcn-1 element (CCCCGCCCACCTCGCGC) does not contain the classical E-box (CACGTG) sequence. There are two predominant ubiquitously expressed TFE3 isoforms (25.Roman C. Cohn L. Calame K. Science. 1991; 254: 94-97Crossref PubMed Scopus (99) Google Scholar). The two transcripts represent differentially spliced mRNAs that yield long, TFE3L, and short, TFE3S, isoforms. The TFE3L isoform is by far the most abundant accounting for more than 80% of the transcripts, and the rest are mostly in the form of TFE3S (25.Roman C. Cohn L. Calame K. Science. 1991; 254: 94-97Crossref PubMed Scopus (99) Google Scholar). There is also another recently described form of TFE3, TFE3-full, that is longer than TFE3L, but its level of expression does not appear to be significant (30.Weterman M.A. Wilbrink M. Geurts van Kessel A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15294-15298Crossref PubMed Scopus (189) Google Scholar, 31.Weterman M.J. van Groningen J.J. Jansen A. van Kessel A.G. Oncogene. 2000; 19: 69-74Crossref PubMed Scopus (70) Google Scholar). TFE3L and TFE3-full contain two activation domains, one acidic (AAD) in the N terminus of the protein and the other proline-rich (Pro-AD) C-terminal to the bHLHzip region (Fig. 1A) (30.Weterman M.A. Wilbrink M. Geurts van Kessel A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15294-15298Crossref PubMed Scopus (189) Google Scholar, 31.Weterman M.J. van Groningen J.J. Jansen A. van Kessel A.G. Oncogene. 2000; 19: 69-74Crossref PubMed Scopus (70) Google Scholar). Although the two activation domains act synergistically, the short spliced variant, TFE3S, lacks the acidic activation domain and its transcriptional activity appears to be diminished (25.Roman C. Cohn L. Calame K. Science. 1991; 254: 94-97Crossref PubMed Scopus (99) Google Scholar). To determine if TFE3 binds to the bcn-1 motif directly, the three forms of the protein,35S-TFE3-full, 35S-TFE3L, and 35S-TFE3S (Fig. 1A) were synthesized in a cell-free system and pull-down assays were done using biotinylated double-stranded oligonucleotide containing the bcn-1 element.35S-Labeled luciferase served as a negative control.35S-Labeled proteins were incubated with biotinylated double-stranded oligonucleotide containing the bcn-1 element, and then the mix was added to streptavidin beads. After binding, beads were washed, and proteins were eluted by boiling in SDS-loading buffer.35S-Labeled translational products (Fig. 1B, lanes 1–4) and 35S-labeled proteins eluted from the beads (Fig. 1B, lanes 5–8) were resolved by SDS-PAGE and were analyzed by densitometry (Fig. 1C). All three isoforms of TFE3 (Fig. 1B, lanes 6–8) bound to the bcn-1 element with apparently similar affinity (Fig. 1C). In contrast, 35S-luciferase did not bind the oligonucleotide at all (lane 5). These results demonstrate that TFE3 binds to DNA directly. The fact that all three TFE3 isoforms bind the bcn-1 element equally well is to be expected, because all three contain an intact bHLHzip domain that has previously been shown to mediate the binding to the E-box (32.Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 33.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (259) Google Scholar). This result suggests that all three isoforms may regulate the activity of the LAMC1 promoter. The bcn-1 motif contains the CAC trinucleotide found in the E-box, suggesting that these nucleotides could mediate TFE3 binding. To test this, we compared binding of 35S-TFE3L to bcn-1 oligonucleotide in the presence of either no competitor, double-stranded oligonucleotide containing wild-type bcn-1, or double-stranded oligonucleotide mutant bcn-1 in which the AC dinucleotide was mutated to GG. As illustrated in Fig. 2, 35S-TFE3 binding to bcn-1 was greatly diminished when wild-type bcn-1 competitor was present (compare lane 3 to 2). In contrast, in the presence of a competitor containing the CAC to CGG mutation, there was little or no decrease in the amount of bcn-1-bound 35S-TFE3 (compare lane 4 to 2). These studies demonstrate that the binding of TFE3 to the bcn-1 element is specific and that the AC dinucleotide found in this motif is important for mediating this interaction. To test if TFE3 can activate the LAMC1promoter and if it is bcn-1-dependent, we carried out reporter gene assays in transient transfections of primary rat mesangial cells. Firefly luciferase reporter gene driven by the LAMC1 promoter was co-expressed with either empty expression vector or expression vector containing cDNA-encoding TFE3L. The β-galactosidase gene driven by the SV40 promoter was used as a control for transfection efficiency. These experiments showed a greater than 3-fold activation of the wild-type −1077/−20LAMC1 promoter fragment (subcloned into pGL3-basic plasmid) by TFE3 compared with expression vector (Fig. 3B, wt-bcn-1). Mutation of the two nucleotides within the bcn-1 element that are key for TFE3 binding (Fig. 2, B and C), decreased both the constitutive activity of the LAMC1 promoter and its response to TFE3 (Fig. 3B, mt-bcn-1). As in the case of TFE3L, expression of TFE3-full also activated (greater than 3-fold induction) the LAMC1 promoter in a bcn-1-dependent manner (data not shown). These results demonstrate that the LAMC1 promoter is responsive to TFE3 and that the bcn-1 element plays an important role in TFE3-mediated response. The transforming growth factor-β (TGF-β) regulates a host of cellular effects, including cell proliferation, development, and differentiation (34.Hill C.S. Cell Signaling. 1996; 8: 533-544Crossref PubMed Scopus (26) Google Scholar). TGF-β activates expression of many genes, including LAMC1 (7.Suzuki H. O'Neill B.C. Suzuki Y. Denisenko O.N. Bomsztyk K. J. Biol. Chem. 1996; 271: 18981-18988Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). TGF-β binds two cell surface serine/threonine kinase receptors, TβRI and TβRII, resulting in the activation of the TβRI kinase, an enzyme that in turn phosphorylates transcription factors such as Smad2 and Smad3 (35.Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar). After binding to Smad4, the Smad2·Smad4 and Smad3·Smad4 complexes translocate to the nucleus where they regulate expression of a diversity of gene targets that account for the pleiotropic effects of TGF-β (35.Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar). Smad proteins are composed of two Mad homology (MH1 and MH2) domains separated by a linker (26.Hua X. Miller Z.A. Wu G. Shi Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13130-13135Crossref PubMed Scopus (134) Google Scholar) (Fig. 4A). Although the function of the MH domains has not been fully defined, in the case of Smad3, the MH1 domain mediates its binding to the Smad-binding element (SBE), AGAC (35.Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar). Mesangial cells express TFE3L (data not shown) and several of the Smad proteins, including Smad2, Smad3, and Smad4 (36.Poncelet A.C. Schnaper H.W. J. Biol. Chem. 2001; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). It has previously been shown that TFE3L and Smad synergistically activate the plasminogen activator inhibitor-1 (PAI-1) promoter (33.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (259) Google Scholar). We wondered if there is similar cooperation between TFE3 and Smad proteins in the activation of the LAMC1 promoter in mesangial cells. Expression of either Smad3 or Smad4 alone increased the activity of the −1077/−20 LAMC1 promoter fragment by more than 2-fold (Fig. 4B). This result shows the LAMC1 promoter is Smad-responsive. When TFE3 was expressed with either Smad3 or Smad4, the activity of the LAMC1 promoter increased by 8- and 10-fold, respectively (Fig. 4B). Co-expression of TFE3, Smad3, and Smad4 did not further increase the promoter activity beyond that seen with TFE3 and one of the two Smads (Fig. 4C). In agreement with the PAI-1 study (33.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (259) Google Scholar), these results demonstrate TFE3·Smad3 cooperative activation of the LAMC1 promoter. Expression of TFE3 and Smad3 in HeLa cells synergistically activated LAMC1 promoter cloned upstream of luciferase gene (data not shown), thus the cooperation between the two transcription factors is not specific to mesangial cells. The sequence AGAC is sufficient for high affinity binding of Smad3 and Smad4 to SBEs (35.Kretzschmar M. Massague J. Curr. Opin. Genet. Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar). Previously it has been shown that two closely located SBEs are required for the TFE3·Smad-med" @default.
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• W2039717963 title "bcn-1 Element-dependent Activation of the Laminin γ1 Chain Gene by the Cooperative Action of Transcription Factor E3 (TFE3) and Smad Proteins" @default.
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