Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Upstream Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Element-1, a Novel and Essential Regulatory DNA Motif in the Human TIMP-1 Gene Promoter, Directly Interacts with a 30-kDa Nuclear Protein

2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês

10.1074/jbc.275.9.6657

ISSN

1083-351X

Autores

Julie E. Trim, Satinder K. Samra, Michael J.P. Arthur, Matthew C. Wright, Martin McAulay, Raj K. Beri, Derek A. Mann,

Tópico(s)

Wnt/β-catenin signaling in development and cancer

Resumo

Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5′-TGTGGTTTCCG-3′) present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states. Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5′-TGTGGTTTCCG-3′) present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states. extracellular matrix tissue inhibitor of metalloproteinases hepatic stellate cell upstream TIMP-1 element-1 chloramphenicol acetyltransferase nuclear factors of activated T cells wild type electrophoretic mobility shift assay The complex interactions between cells and components of their surrounding extracellular matrix (ECM)1 are critical for many cellular properties including division, migration, differentiation, and death (1.Lukashev M.E. Werb Z. Trends Cell Biol. 1998; 8: 437-441Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Remodeling of the ECM occurs during embryonic development and during certain normal physiological processes such as wound healing (2.Werb Z. Chin J.R. Ann. N. Y. Acad. Sci. 1998; 857: 110-118Crossref PubMed Scopus (174) Google Scholar, 3.Raghow R. FASEB J. 1994; 8: 823-831Crossref PubMed Scopus (366) Google Scholar). However, restructuring of the ECM has also been implicated in various human pathologies including impaired wound healing and tissue fibrosis (4.Friedman S. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar), rheumatoid arthritis (5.Cawston T. Mol. Med. Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), restenosis following balloon angioplasty (6.Batchelor W.B. Robinson R. Strauss B.H. Prog. Cardiovasc. Dis. 1998; 41: 35-49Crossref PubMed Scopus (36) Google Scholar), atherosclerosis (7.Newby A.C. Zaltsman A.B. Cardiovasc. Res. 1999; 41: 345-360Crossref PubMed Scopus (274) Google Scholar), and tumor development, invasion and metastasis (1.Lukashev M.E. Werb Z. Trends Cell Biol. 1998; 8: 437-441Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 8.Sternlicht M.D. Lochter A. Sympson C.J. Huey B. Rougier J-P. Gray J.W. Pinkel D. Bissel M.J. Werb Z. Cell. 1999; 98: 137-146Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar). Under normal physiological conditions, a fine balance of ECM-degrading matrix metalloproteinases and their naturally occurring inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), controls the rate of ECM turnover (5.Cawston T. Mol. Med. Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9.Edwards D.R. Beaudry P.P. Laing T.D. Kowal V. Leco K.J. Leco P.A. Lim M.S. J. Obes. Relat. Metab. Disord. 1996; 20 Suppl. 3: 9-15Google Scholar, 10.Reynolds J.J. Oral Dis. 1996; 2: 70-76Crossref PubMed Scopus (179) Google Scholar, 11.Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar). Since the TIMP family of protease inhibitors (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) are key regulators of matrix metalloproteinase activity and therefore ECM degradation (11.Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar), there is great interest in understanding the cellular mechanisms that control their expression. The prototypic member of the TIMP family, TIMP-1, is of particular interest, since, in addition to its classical role as a broad specific inhibitor of matrix metalloproteinases (12.Denhardt D.T. Feng B. Edwards D.R. Cocuzzi E.T. Malyankar U.M. Pharmacol. Ther. 1993; 59: 329-341Crossref PubMed Scopus (266) Google Scholar), TIMP-1 has also been reported to possess growth factor-like (13.Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iawata K. FEBS Lett. 1992; 298: 29-32Crossref PubMed Scopus (645) Google Scholar, 14.Hayakawa T. Yamashita K. Kishi J. Harigaya K. FEBS Lett. 1990; 268: 125-128Crossref PubMed Scopus (48) Google Scholar) and anti-apoptotic properties (15.Alexander C.M. Howard E.W. Bissel M.J. Werb Z. J. Cell Biol. 1996; 135 (1667): 1669Crossref PubMed Scopus (203) Google Scholar, 16.Boudreau N. Sympson C.J. Werb Z. Bissel M. Science. 1995; 267: 891-893Crossref PubMed Scopus (1114) Google Scholar, 17.Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (361) Google Scholar, 18.Guedez L. Courtemanch L. Stetler-Stevenson M. Blood. 1998; 92: 1342-1349Crossref PubMed Google Scholar). Guedez et al. (17.Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (361) Google Scholar) recently showed that TIMP-1 inhibits apoptosis and promotes survival of Burkitts' lymphoma cell lines. These observations support work implicating TIMP-1 in cancer, tumor invasion, metastasis, and angiogenesis (19.Kossakowska E.E. Urbanski S.J. Edwards D.R. Blood. 1991; 77: 2475-2481Crossref PubMed Google Scholar, 20.Mansoor A. Birkedal-Hansen B. Lim M.S. Guedez L. Stetler-Stevenson W.G. Stetler-Stevenson M. Mod. Pathol. 1997; 10: 130AGoogle Scholar, 21.Mimori K. Mori M. Shiraishi T. Fujie T. Baba K. Haraguchi M. Abe R. Ueo H. Akiyoshi T. Br. J. Cancer. 1997; 76: 531-536Crossref PubMed Scopus (99) Google Scholar, 22.Ree A.H. Florenes V.A. Berg J.P. Maelandsmo G.M. Nestland J.M. Fostad O. Clin. Cancer Res. 1997; 3: 1623-1628PubMed Google Scholar, 23.Stetler-Stevenson M. Mansoor A. Lim M. Fukushima P. Kehrl J. Marti G. Ptaszynski K. Wang J. Stetler-Stevenson W.G. Blood. 1997; 89: 1708-1715Crossref PubMed Google Scholar). TIMP-1 gene transcription and mRNA expression can be stimulated by a wide variety of agents including serum, growth factors, phorbol esters, cytokines, and viruses (24.Gewart D.R. Coulombe B. Castelino M. Skup D. Williams B.R.G. EMBO J. 1987; 6: 651-657Crossref PubMed Scopus (58) Google Scholar, 25.Coulombe B. Ponton A. Daigneault L. Williams B.R.G. Skup D. Mol. Cell. Biol. 1988; 8: 3227-3234Crossref PubMed Scopus (34) Google Scholar, 26.Campbell C.E. Flenniken A.M. Skup D. Williams B.R.G. J. Biol. Chem. 1991; 266: 7199-7206Abstract Full Text PDF PubMed Google Scholar, 27.Edwards D.R. Rocheleau H. Sharma R.R. Wills A.J. Cowie A. Hassell J.A. Heath J.K. Biochim. Biophys. Acta. 1992; 1171: 41-55Crossref PubMed Scopus (129) Google Scholar, 28.Uchijima M. Sato H. Fuji M. Seiki M. J. Biol. Chem. 1994; 269: 14946-14950Abstract Full Text PDF PubMed Google Scholar, 29.Bugno M. Graeve L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordulla T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar). Activation of hepatic stellate cells (HSCs), a key event in the pathophysiology of liver fibrosis (4.Friedman S. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar), is also accompanied by induction of TIMP-1 promoter activity and mRNA expression (30.Iredale J.P. Benyon R.C. Arthur M.J.P. Ferris W.F. Alcolado R. Winwood P.J. Clark N. Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar, 31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). Nuclear run-on transcription assays employed in some of these studies demonstrate that induction occurs primarily at the level of gene transcription, although TIMP-1 expression in U937 cells appears to be regulated at least in part by changes in mRNA stability (32.Doyle G.A.R. SaarialhoKere U.K. Parks W.C. Biochemistry. 1997; 36: 2492-2500Crossref PubMed Scopus (13) Google Scholar). Structural features of the human, murine, and rat TIMP-1 gene promoters have been described previously (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). All three promoters lack a classical TATA box and contain an evolutionary conserved 22-bp serum response element located 75 bp upstream of the major transcription start site (24.Gewart D.R. Coulombe B. Castelino M. Skup D. Williams B.R.G. EMBO J. 1987; 6: 651-657Crossref PubMed Scopus (58) Google Scholar, 26.Campbell C.E. Flenniken A.M. Skup D. Williams B.R.G. J. Biol. Chem. 1991; 266: 7199-7206Abstract Full Text PDF PubMed Google Scholar, 29.Bugno M. Graeve L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordulla T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar, 31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 33.Clark I.M. Rowan A.D. Edwards D.R. Mann D.A. Bahr M.J. Cawston T.E. Biochem. J. 1997; 324: 611-617Crossref PubMed Scopus (79) Google Scholar). The serum response element, which is critical for responsiveness of the promoter to growth factors, cytokines, and viruses, is composed of binding sites for AP-1 (Fos/Jun), signal transducer and activator of transcription, and Pea3 (Ets) transcription factors. Mutagenesis studies performed in several primary and transformed cell lines have established that the AP-1 site within the serum response element is critical for basal and inducible transcription (26.Campbell C.E. Flenniken A.M. Skup D. Williams B.R.G. J. Biol. Chem. 1991; 266: 7199-7206Abstract Full Text PDF PubMed Google Scholar, 27.Edwards D.R. Rocheleau H. Sharma R.R. Wills A.J. Cowie A. Hassell J.A. Heath J.K. Biochim. Biophys. Acta. 1992; 1171: 41-55Crossref PubMed Scopus (129) Google Scholar, 29.Bugno M. Graeve L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordulla T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar, 33.Clark I.M. Rowan A.D. Edwards D.R. Mann D.A. Bahr M.J. Cawston T.E. Biochem. J. 1997; 324: 611-617Crossref PubMed Scopus (79) Google Scholar, 34.Logan S.K. Garabedian M.J. Campbell C.E. Werb Z. J. Biol. Chem. 1996; 271: 774-782Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). We have recently reported that this AP-1 site is critical for the function of a minimal active 162-bp human TIMP-1 promoter in human fibroblasts and culture-activated rat HSCs (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 33.Clark I.M. Rowan A.D. Edwards D.R. Mann D.A. Bahr M.J. Cawston T.E. Biochem. J. 1997; 324: 611-617Crossref PubMed Scopus (79) Google Scholar). The molecular events underlying HSC activation and induction of TIMP-1 can be studied in a cell culture model in which freshly isolated primary HSCs cultured for several days on tissue culture plastic undergo a similar phenotypic transformation to that observedin vivo (4.Friedman S. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar, 30.Iredale J.P. Benyon R.C. Arthur M.J.P. Ferris W.F. Alcolado R. Winwood P.J. Clark N. Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar, 31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). Studies using this culture model showed that induction of TIMP-1 promoter activity occurred at culture time points (5 days or more) associated with morphological and biochemical activation of rat HSCs and with induced expression of JunD, Fra2, and FosB, implicating these AP-1 proteins in the control of TIMP-1 expression in activated HSCs (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). Whereas the role of the AP-1 containing serum response element site in the regulation of TIMP-1 gene transcription is well characterized, much of the 162-bp minimal active human TIMP-1 promoter remains functionally undefined. We have therefore used DNase I footprinting (35.Jackson S.P. James B.D. Higgens S.J. Gene Transcription: A Practical Approach. Oxford University Press, Oxford1993: 189-242Google Scholar) to identify DNA-protein interactions occurring on the promoter in cells expressing high levels of TIMP-1. We report the identification of previously undescribed DNA-protein interactions at a novel regulatory sequence named upstream TIMP-1 element 1 (UTE-1). The site appears to be essential for transcriptional activity in a variety of cell types including activated primary human and rat HSCs as well as transformed human cell lines. We describe the characterization of nuclear proteins capable of interacting with the UTE-1 sequence in a specific manner and the identification of a 30-kDa species that directly binds to UTE-1 DNA. HSCs were isolated from the livers of normal Harlan Sprague Dawley rats (400 ± 50 g) by sequential perfusion with pronase and collagenase as described previously (36.Arthur M.J.P. Friedman S.L. Roll F.J. Bissell D.M. J. Clin. Invest. 1992; 84: 1076-1085Crossref Scopus (155) Google Scholar). HSCs were separated from the cell suspension over an 11.5% Optiprep (Nycomed Pharma AS, Oslo) gradient followed by elutriation. HSCs were seeded onto plastic and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 16% fetal calf serum (Life Technologies) and maintained at 37 °C in an atmosphere of 5% CO2. Human HSCs were isolated from the livers of adult male patients following partial hepatectomy. Sequential perfusion with pronase and collagenase was carried out as described for isolation of rat HSCs (36.Arthur M.J.P. Friedman S.L. Roll F.J. Bissell D.M. J. Clin. Invest. 1992; 84: 1076-1085Crossref Scopus (155) Google Scholar). HSCs were separated from the cell suspension over an 11.5% Optiprep gradient and cultured onto plastic in Dulbecco's modified Eagle's medium supplemented with 16% fetal calf serum and maintained at 37 °C in an atmosphere of 5% CO2. Nuclear extracts were prepared from cells using a modified version of the protocol described by Dignamet al. (37.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9149) Google Scholar). In brief, cells (107) were harvested into 5 ml of ice-cold phosphate-buffered saline by centrifugation at 2500 rpm. The pellets were resuspended into 100 μl of Dignam buffer A containing 0.2% Nonidet P-40 and 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride. The lysate was centrifuged at 8000 rpm for 10 s to pellet the Nonidet P-40-insoluble material, and the supernatant was removed. The pellet was resuspended into 50 μl of Dignam buffer C, incubated on ice for 10 min with occasional vortexing to disrupt the nuclear membranes. Extracts were centrifuged for 1 min at 8000 rpm, the supernatants were removed, and the pellets were discarded. The protein content of the nuclear extracts was determined using the Bio-Rad DC assay kit (Bio-Rad). The TIMP-1 minimal promoter (positions −102 to +60) was 5′-end-labeled with [α-32P]dATP (3000 Ci/mmol, Amersham Pharmacia Biotech) using avian myeloblastosis virus reverse transcriptase (10 units; Promega). Nuclear extract (10 μg) from HSCs was incubated with 1 μg of poly(dI-dC) nonspecific DNA competitor (Sigma) for 10 min, and the radiolabeled TIMP-1 minimal promoter (100 ng) was then added and incubated for a further 20 min. DNase I (0.05 units; type 4, bovine pancreas; Sigma) was added to the reaction mixture and incubated for 1 min. All reactions were carried out at 4 °C. DNase I footprinting reactions were resolved on a 8% denaturing polyacrylamide gel alongside a purine marker ladder. A 20-base pair oligonucleotide (UTE-1) was constructed to the region of the minimal promoter protected from DNase I digestion in the DNase I footprinting experiments. The UTE-1 sense strand oligonucleotide (5′-AGGCCTGTGGTTTCCGCACC-3′) was 5′-end-labeled with [γ-32P]ATP (3000 Ci mmol−1; Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Amersham Pharmacia Biotech). The labeled UTE-1 sense strand was then annealed to the UTE-1 antisense strand (5′-GGTGCGGAAACCACAGGCCT-3′). EMSA reactions were performed as described previously (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 38.Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (121) Google Scholar). The standard EMSA reaction consisted of an initial incubation of the nuclear extract (5 μg) with 1 μg of poly(dI-dC) in a total volume of 18 μl for 10 min. The annealed radiolabeled oligonucleotide probe (2 μl; 0.1 ng μl−1) was added to the reaction and incubated for a further 20 min. All reactions were carried out at 4 °C. For competition assays excess unlabeled oligonucleotide (2–20 ng; Table I) was added to the reaction mixture with the nuclear extract and poly(dI-dC) for the initial incubation, prior to the addition the probe. EMSA reaction mixtures were resolved by electrophoresis on an 8% nondenaturing polyacrylamide gel (37:5:1).Table IThe sequences of oligonucleotides used in competition EMSA experimentsOligonucleotideSequenceUTE-1 wild type (wt)5′-AGGCCTGTGGTTTCCGCACC-3′UTE-1 mutant (M)5′-AGGCCCAGTAGCTCCACACC-3′UTE-1 large scan 1 (ls1)5′-CATTTTGTGGTTTCCGCACC-3′UTE-1 large scan 2 (ls2)5′-AGGTTCAGGGTTTCCGCACC-3′UTE-1 large scan 3 (ls3)5′-AGGCCCAGTATTTCCGCACC-3′UTE-1 large scan 4 (ls4)5′-AGGCCTGGTAGCTCCGCACC-3′UTE-1 large scan 5 (ls5)5′-AGGCCTGTGAGCTCCGCACC-3′UTE-1 large scan 6 (ls6)5′-AGGCCTGTGGTCTCCACACC-3′UTE-1 large scan 7 (ls7)5′-AGGCCTGTGGTTTCCAAGTT-3′UTE-1 point scan 1 (ps1)5′-AGGCCCATGGTTTCCGCACC-3′UTE-1 point scan 2 (ps2)5′-AGGCCTAGGGTTTCCGCACC-3′UTE-1 point scan 3 (ps3)5′-AGGCCTGGTGTTTCCGCACC-3′UTE-1 point scan 4 (ps4)5′-AGGCCTGTTATTTCCGCACC-3′UTE-1 point scan 5 (ps5)5′-AGGCCTGTGAGTTCCGCACC-3′UTE-1 point scan 6 (ps6)5′-AGGCCTGTGGGCTCCGCACC-3′AP35′-TACTGGGACTTTCCACA-3′AP3-like5′-CAGTGTGGAAAATCTCT-3′NFAT5′-GAAAGGAGGAAAAACTG-3′The UTE-1 sequence was determined from a footprinted region of the TIMP-1 minimal promoter. Mutations (5 or 2 base pairs, as underlined) were introduced across the region to determine the essential binding nucleotides for this novel site. Open table in a new tab The UTE-1 sequence was determined from a footprinted region of the TIMP-1 minimal promoter. Mutations (5 or 2 base pairs, as underlined) were introduced across the region to determine the essential binding nucleotides for this novel site. A CAT reporter plasmid (pBLCAT3) containing the TIMP-1 162-bp minimal promoter (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar) cloned into the HindIII andPstI site, upstream of the CAT gene, was used in all functional studies. The UTE-1 mutant sequence was generated by polymerase chain reaction-mediated mutagenesis of the wild type minimal promoter using the following oligonucleotide primers: mutant forward primer (5′-CCTGGAGGCCCAGTAGCTCCACACCCGCTG-3′) and reverse primer (5′- CAAGCTGCAGCCCAGCTCCGGTCCCTGCTG-3′). Primary human and rat HSCs (8-day activated) seeded at 1 × 106/well on a six-well plate and Jurkat T cells (5 × 106) were transfected using 1 μg of purified plasmid DNA (Qiagen Maxiprep, Qiagen) with the transfection reagent Effectene (Qiagen) according to the manufacturer's instructions and left in contact with the cells for 48 h. CAT assays were performed as described by Gorman (39.Gorman C. Glover D.M. DNA Cloning. 2. IRL Press, Oxford1985: 143-190Google Scholar). In brief, cell extracts were prepared by repeated freeze-thaw cycles and normalized for protein content using the Bio-Rad DC protein assay, and CAT activities were determined using [14C]chloramphenicol (Amersham Pharmacia Biotech) and acetyl-CoA (Sigma). Acetylated products were separated by thin layer chromatography and quantitated by phosphor imaging. CAT activities were normalized to the amount of DNA taken up by cultures, determined using a modification of Hirt's assay (40.Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3345) Google Scholar). The UTE-1 oligonucleotide was radiolabeled as described previously for EMSA. Nuclear extracts (10 μg) were incubated with 2 μg of poly(dI-dC) in a total volume of 8 μl for 10 min; the oligonucleotide probe (2 μl; 0.1 ng μl−1) was then added to the reaction and incubated for a further 20 min. DNA-protein complexes were cross-linked by UV radiation for 1 h at 1200 KJ cm−2 (λ = 305 nm) and resolved by electrophoresis on 12.5% SDS-polyacrylamide gel (20 mA, 1 h). All reactions were carried out at 4 °C. The Southwestern blotting technique is a variation of the traditional Western blotting method (35.Jackson S.P. James B.D. Higgens S.J. Gene Transcription: A Practical Approach. Oxford University Press, Oxford1993: 189-242Google Scholar). Following transfer of proteins onto a nitrocellulose membrane DNA binding proteins are detected with a radiolabeled DNA probe (UTE-1 oligonucleotide). Nuclear extracts (30 μg) from rat activated HSCs, Jurkat T cells, and Daudi B cells were separated on a 12.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The UTE-1 DNA probe (15 ng) was prepared as described for EMSA studies and hybridized to the membrane at room temperature for 30 min. Potential binding sites for transcription factors within the TIMP-1 minimal promoter were identified by DNase I footprinting. Nuclear extract from 1-day and 8-day cultured rat HSCs were used for footprinting in an attempt to identify DNA-protein interactions that are induced during HSC activation. When using nuclear extract from activated 8-day cultured HSCs, a large proportion of the TIMP-1 minimal promoter was protected from the DNase I digestion in comparison with the control track (−) digested in the absence of nuclear extract (Fig. 1 A). The protected regions included the potential Sp1 site, TATA-like box, and a previously undescribed 11-bp region downstream from the AP-1 and Pea-3 sites at nucleotides −63 to −53 of the human TIMP-1 gene (Fig.1 B). The 11-bp region was also protected from DNase I digestion by nuclear proteins isolated from 1-day cultured HSCs, although the intensity of the footprint was markedly reduced relative to that observed with 8-day cultured HSCs (Fig. 1 A). The protected 11-bp sequence (5′-TGTGGTTTCCG-3′) was analyzed on sequence data bases (EMBL, GenBankTM, Transfac) in an attempt to identify possible matches with previously characterized transcription factor binding sites. Some sequence homology was found with the AP3 site (5′-TGGGACTTTCCA-3′) and an AP3-like (AP3-L; 5′-TGTGGAAAATCT-3′) site that resembles the nuclear factors of activated T cells (NFAT-1; 5′-AGGAGGAAAAACT-3′) binding site (41.Van Lint C. Amella C.A. Emiliani S. John M. Jie T. Verdin E. J. Virol. 1997; 71: 6113-6127Crossref PubMed Google Scholar). No exact match was identified; therefore, the footprinted region was named upstream TIMP-1 Element 1 (UTE-1). UTE-1-protein interactions were investigated using a double-stranded 20-base pair oligonucleotide (5′-AGGCCTGTGGTTTCCGCACC-3′), constructed to span the UTE-1 element of the TIMP-1 minimal promoter (TableI). Since TIMP-1 is expressed by a variety of different cell types, it was of interest to establish the expression pattern of UTE-1-binding proteins in various mammalian cell lines as well as primary human and rat HSCs. Fig.2 A shows that a retardation complex was obtained with nuclear extracts from all cell types tested, although there were distinct qualitative and quantitative differences observed between the cells. Culture-activated primary rat HSCs and human Jurkat T cells gave rise to similar high mobility UTE-1 DNA-protein complexes, although the relative abundance was far greater for Jurkat T cells. Culture-activated primary human HSCs assembled a lower mobility complex that was of similar mobility and abundance to UTE-1 complexes observed with Daudi B cell and THP-1 monocyte-macrophage cell lines. Passaging of human HSCs, which gives a higher selectivity for HSCs over other cell types (Kupfer cells in particular) that contaminate the original primary culture, was associated with a change in the mobility and abundance of the UTE-1 binding complex. These changes generated a DNA-protein complex that was more similar to the complex observed with pure (95%) primary rat HSCs. These results suggest that UTE-1-binding proteins probably exist in most mammalian cell types; however, the nature and abundance of the DNA-protein complexes formed at promoters may vary between cell types. Since we have previously reported that transcriptional activity of the human TIMP-1 promoter is induced during HSC activation (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar) it was of interest to determine if UTE-1-binding proteins are also induced during the culturing of freshly isolated HSCs. For these experiments, we used rat HSCs previously shown to support transcriptional activity of the human TIMP-1 promoter (31.Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). EMSA reactions with nuclear extracts from different time points of HSC activation on plastic revealed the existence of multiple DNA-protein complexes of dissimilar electrophoretic mobilities (Fig. 2 B). Freshly isolated HSCs expressed multiple UTE-1 complexes that upon culturing were replaced by two complexes of lower mobility that persisted for the first 24 h of culture. Subsequent periods of culture were associated with the loss of the low mobility complexes and induction of at least one higher mobility complex that was maximally expressed at day 5 of culture and persisted for at least a further 9 days (data not shown). It is noteworthy that the appearance of the high mobility UTE-1 complex at 3 days and its maximal induction at 5 days are synchronous with thede novo expression and peak induction of TIMP-1 mRNA, respectively (30.Iredale J.P. Benyon R.C. Arthur M.J.P. Ferris W.F. Alcolado R. Winwood P.J. Clark N. Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar). Sequence specificity of the UTE-1 DNA-protein interaction was tested by competition EMSA reactions. A 50-fold excess of unlabeled wild type (wt) double-stranded oligonucleotide competed out all protein binding to the radiolabeled UTE-1 site using nuclear extracts from activated rat HSCs (Fig. 3 A). Hence, binding of nuclear proteins to the UTE-1 sequence is s

Referência(s)