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• W2067982008 abstract "The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin α5β1. In this work, we determined whether the activity directed by the α5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/α5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the α5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3′ half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its α5β1 integrin activates a signal transduction pathway that results in the transcriptional activation of the α5 gene likely through altering the phosphorylation state of Sp1. The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin α5β1. In this work, we determined whether the activity directed by the α5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/α5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the α5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3′ half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its α5β1 integrin activates a signal transduction pathway that results in the transcriptional activation of the α5 gene likely through altering the phosphorylation state of Sp1. extracellular matrix fibronectin rabbit corneal epithelial cells fibronectin-responsive element electrophoretic mobility shift assays chloramphenicol acetyltransferase mitogen-activated protein kinases extracellular signal-regulated kinase MAPK/ERK kinase human growth hormone base pair oncostatin M low density lipoprotein receptor vascular endothelial growth factor acetylcholine receptor fetal bovine serum Corneal wounds account for a substantial proportion of all visual disabilities and medical consultations for ocular problems in North America. They can be superficial with damage limited to the epithelium or associated with a deeper involvement of the epithelial basement membrane and of the stromal lamella. Severe recurrent and persistent corneal wounds are most commonly secondary to ocular diseases and damage such as recurrent erosion, mild chemical burns, superficial herpetic infections, neuroparalytic cornea, autoimmune diseases, and stromal ulcerations due to viral or bacterial infections or to severe burns (1Reim M. Kottek A. Schrage N. Prog. Retin. Eye Res. 1997; 16: 183-225Crossref Scopus (47) Google Scholar). Despite currently available treatments, many of these corneal wounds persist for weeks and months or else recur frequently and can progress to corneal perforation. Tissue repair requires cell migration, proliferation, and adhesion. Cell adhesion and migration in turn require extracellular matrix (ECM)1 synthesis and assembly. ECM is a complex, cross-linked structure of proteins and polysaccharides. It organizes the geometry of normal tissues. Fibronectin (FN) is an ECM adhesion protein identified as a potential wound healing agent because of its cell attachment, migration, differentiation, and orientation properties (for a review see Refs. 2Humphries M.J. Obara M. Olden K. Yamada K.M. Cancer Invest. 1989; 7: 373-393Crossref PubMed Scopus (99) Google Scholar, 3Hynes R.O. Sci. Am. 1986; 254: 42-51Crossref PubMed Scopus (166) Google Scholar, 4Ruoslahti E. Annu. Rev. Biochem. 1988; 57: 375-413Crossref PubMed Scopus (1058) Google Scholar). In the unwounded rat eye, FN is observed by immunohistological staining at the level of the corneal epithelium basement membrane (5Murakami J. Nishida T. Otori T. J. Lab. Clin. Med. 1992; 120: 86-93PubMed Google Scholar, 6Sramek S.J. Wallow I.H. Bindley C. Sterken G. Invest. Ophthalmol. & Visual Sci. 1987; 28: 500-505PubMed Google Scholar, 7Tuori A. Burgeson R.E. Terttunen J. Virtanen I. Uusitalo H. Cornea. 1996; 15: 286-294Crossref PubMed Scopus (101) Google Scholar). Shortly after corneal injury, the basal cells that border the injured area and stromal keratocytes start producing massive amounts of FN (5Murakami J. Nishida T. Otori T. J. Lab. Clin. Med. 1992; 120: 86-93PubMed Google Scholar, 8Araki K. Ohashi Y. Kinoshita S. Hayashi K. Kuwayama Y. Tano Y. Curr. Eye Res. 1994; 13: 203-211Crossref PubMed Scopus (101) Google Scholar, 9Cai X. Foster C.S. Liu J.J. Kupferman A.E. Filipec M. Colvin R.B. Lee S.J. Invest. Ophthalmol. & Visual Sci. 1993; 34: 3585-3592PubMed Google Scholar, 10Espaillat A. Lee S.J. Arrunategui-Correa V. Foster C.S. Vitale A. DiMeo S. Colvin R.B. Curr. Eye Res. 1994; 13: 325-330Crossref PubMed Scopus (10) Google Scholar, 11Nakamura M. Sato N. Chikama T.I. Hasegawa Y. Nishida T. Exp. Eye Res. 1997; 64: 1043-1050Crossref PubMed Scopus (53) Google Scholar). FN promotes corneal cell migration both in vivo (12Gundorova R.A. Brikman I.V. Ibadova S.I. Issaeva R.T. Eur. J. Ophthalmol. 1994; 4: 202-210Crossref PubMed Scopus (10) Google Scholar, 13Nishida T. Nakagawa S. Watanabe K. Yamada K.M. Otori T. Berman M.B. Invest. Ophthalmol. & Visual Sci. 1988; 29: 1820-1825PubMed Google Scholar) and in vitro (14Mooradian D.L. McCarthy J.B. Skubitz A.P. Cameron J.D. Furcht L.T. Invest. Ophthalmol. & Visual Sci. 1993; 34: 153-164PubMed Google Scholar) by acting as a temporary extracellular matrix to which corneal epithelial cells attach as they migrate over the wounded area (13Nishida T. Nakagawa S. Watanabe K. Yamada K.M. Otori T. Berman M.B. Invest. Ophthalmol. & Visual Sci. 1988; 29: 1820-1825PubMed Google Scholar, 15Nishida T. Nakamura M. Mishima H. Otori T. J. Cell. Physiol. 1990; 145: 549-554Crossref PubMed Scopus (66) Google Scholar). Once the wound is re-epithelialized, the subepithelial immunohistological staining of FN progressively decreases (5Murakami J. Nishida T. Otori T. J. Lab. Clin. Med. 1992; 120: 86-93PubMed Google Scholar, 16Fujikawa L.S. Foster C.S. Harrist T.J. Lanigan J.M. Colvin R.B. Lab. Invest. 1981; 45: 120-129PubMed Google Scholar, 17Fujikawa L.S. Foster C.S. Gipson I.K. Colvin R.B. J. Cell Biol. 1984; 98: 128-138Crossref PubMed Scopus (155) Google Scholar, 18Saika S. Kobata S. Hashizume N. Okada Y. Yamanaka O. Cornea. 1993; 12: 383-390Crossref PubMed Scopus (52) Google Scholar). The increase in FN expression that has been reported to occur during corneal wound healing was postulated to be coordinated with the expression of its major integrin receptor α5β1 (5Murakami J. Nishida T. Otori T. J. Lab. Clin. Med. 1992; 120: 86-93PubMed Google Scholar), as has also been shown for laminin and tenascin and their corresponding integrin receptor subunits α6 and α9, respectively (19Garana R.M. Petroll W.M. Chen W.T. Herman I.M. Barry P. Andrews P. Cavanagh H.D. Jester J.V. Invest. Ophthalmol. & Visual Sci. 1992; 33: 3271-3282PubMed Google Scholar, 20Stepp M.A. Zhu L. Cranfill R. Invest. Ophthalmol. & Visual Sci. 1996; 37: 1593-1601PubMed Google Scholar, 21Stepp M.A. Zhu L. J. Histochem. Cytochem. 1997; 2: 189-201Crossref Scopus (68) Google Scholar). For instance, the integrin α5β1 was shown to be present during corneal wound healing after radial keratectomy (22Zhu L. Stepp M.A. Invest. Ophthalmol. & Visual Sci. 1996; 37: 1011Google Scholar). Direct evidence that FN can positively alter α5β1 integrin expression at both the protein and mRNA levels has been provided through FN antisense expression studies performed in the epithelium-derived human colon carcinoma cell line Moser (23Rajagopal S. Huang S. Albitar M. Chakrabarty S. J. Cell. Physiol. 1997; 170: 138-144Crossref PubMed Scopus (14) Google Scholar) as well as in murine AKR-2B fibroblasts (24Huang S. Varani J. Chakrabarty S. J. Cell. Physiol. 1994; 161: 470-482Crossref PubMed Scopus (19) Google Scholar). Other indirect evidence linking expression of α5β1 to that of FN has also emerged from recent studies (25Dalton S.L. Marcantonio E.E. Assoian R.K. J. Biol. Chem. 1992; 267: 8186-8191Abstract Full Text PDF PubMed Google Scholar, 26Omigbodun A. Coukos G. Ziolkiewicz P. Wang C.L. Coutifaris C. Endocrinology. 1998; 139: 2190-2193Crossref PubMed Google Scholar). As a consequence, it is not surprising that ECM, through its interactions with membrane-bound integrins, exerts profound influences on the major cellular program of growth, differentiation, and apoptosis by altering, through a number of signal transduction pathways, the transcription of genes whose specific functions are linked to these cellular functions. Binding of ECM components, such as FN, with their corresponding integrin receptors will trigger the activation of intracellular signaling mediators such as focal adhesion kinase, mitogen-activated protein kinases (MAPKs), and Rho family GTPases (for a review see Ref. 27Boudreau N.J. Jones P.L. Biochem. J. 1999; 339: 481-488Crossref PubMed Scopus (517) Google Scholar). Activation of the MAPK signal transduction pathway is of particular interest since it links integrin-mediated signaling to transcriptional regulation of genes that are crucial for cell growth and differentiation. The results presented hereby provided evidence that, by acting on α5 gene expression, such a route of signal transduction might alter cell adhesion properties as well. The downstream cascade of family members that are activated following transient activation of Ras GTP-binding proteins through receptor tyrosine kinases include MAPK/ERK kinase (designated MEK) and ERK1 (p44)/ERK2 (p42) (28Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). Activation of ERK1/ERK2 through phosphorylation causes their translocation to the nucleus, where they have been reported to phosphorylate and activate distinct transcription factors, such as ELK, c-Jun, and c-Myc (29Fukunaga R. Hunter T. EMBO J. 1998; 16: 1921-1933Crossref Scopus (555) Google Scholar, 30Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1163) Google Scholar, 31Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (880) Google Scholar), as well as members of the ETS family (such as PEA3) (32O'Hagan R.C. Tozer R.G. Symons M. McCormick F. Hassell J.A. Oncogene. 1996; 13: 1323-1333PubMed Google Scholar). In the present study, we demonstrated that FN can alter the transcription of the α5 integrin subunit gene at the promoter level. Such a FN responsiveness was shown to be determined by the binding of the transcription factor Sp1 to a target site that is part of a perfect inverted repeat which, by itself, can confer FN responsiveness to an otherwise unresponsive heterologous promoter. Most of all, the FN-activated, integrin-mediated signal transduction pathway appears to require activation of ERK1/ERK2 since the Sp1 DNA binding affinity, and, as a consequence, the FN responsiveness of the α5 promoter were both found to be diminished by blocking their activation with the MEK kinase inhibitor PD98059. Together, these results demonstrate the novel finding that the α5integrin subunit, through activation of the MAPK pathway, can autoregulate its own synthesis in a manner that is dependent on the extracellular concentration of FN. Rabbit corneal epithelial cells (RCECs) were obtained from the central area of freshly dissected rabbit corneas as described previously (33Boisjoly H.M. Laplante C. Bernatchez S.F. Salesse C. Giasson M. Joly M.-C. Exp. Eye Res. 1993; 57: 293-300Crossref PubMed Scopus (50) Google Scholar) and then grown to low (near 15% coverage of the plates), intermediate (near 75% coverage), or high cell density (100% coverage for more than 48 h) under 5% CO2 in SHEM medium supplemented with 5% FBS and 20 μg/ml gentamicin. When indicated, human plasma FN (obtained as described previously (34Regnault V. Rivat C. Geshier C. Stoltz J.F. Rev. Fr. Transfus. Hemobiol. 1990; 33: 391-405PubMed Google Scholar)) or ECM gel (basement membrane matrice from Engelbreth-Holm-Swarm mouse sarcoma, Fisher) was coated for 18 h at 37 °C on the culture dishes at varying concentrations (FN, 1–16 μg per cm2; ECM, 10 μg per cm2). Coated Petri dishes were washed twice with phosphate-buffered saline and blocked at 37 °C with 2% bovine serum albumin in phosphate-buffered saline. Cells were then seeded and grown as above. Inhibition of ERK1/ERK2 was performed by culturing subconfluent RCEC in the presence of 10 μm of the MEK/kinase inhibitor PD98059 (Sigma) for 48 h before cells were harvested. Drosophila Schneider cells (ATCC CRL-1963) were cultured at 28 °C without CO2in Schneider medium (Sigma) supplemented with 10% FBS and 20 μg/ml gentamicin. The plasmids α5−41, α5−92, α5−178, and α5−954, which all bear the chloramphenicol acetyltransferase (CAT) reporter gene fused to DNA fragments from the human α5 gene upstream regulatory sequence extending up to 5′ positions −41, −92, −178, and −954, respectively, but all sharing a common 3′ end located at position +23, have been described previously (35Birkenmeier T.M. McQuillan J.J. Boedeker E.D. Argraves W.S. Ruoslahti E. Dean D.C. J. Biol. Chem. 1991; 266: 20544-20549Abstract Full Text PDF PubMed Google Scholar). The recombinant plasmids bearing one or two sense copies of either the α5 FRE or its mutant derivatives were created by inserting the corresponding double-stranded oligomers upstream from the basal promoter of the mouse p12 gene (into the uniqueBamHI site) that has been previously mutated into its Sp1-binding site (and designated p12.108/M (36Robidoux S. Gosselin P. Harvey M. Leclerc S. Guérin S.L. Mol. Cell. Biol. 1992; 12: 3796-3806Crossref PubMed Scopus (37) Google Scholar)). The Sp1 expression vector pPacSp1 was generously provided by Dr. Guntram Suske (Institute für Molecular Biology und Tumorforschung, Philipps Universität Marburg, Germany), whereas the LacZ expression plasmid pAC5/V5-His/LacZ was obtained from Invitrogen (Carlsbad, CA). The double-stranded oligonucleotides used in the present study were chemically synthesized using a Biosearch 8700 apparatus (Millipore). They contained the DNA sequence from the human α5promoter comprised between positions −82 and −56 and designated the α5 FRE (5′-GATCAGCCGGGAGTTTGGCAAACTCCTCCCC-3′) or its mutated derivatives (α5FRE/m5′, 5′-GATCAGCCGAAAAATTGGCAAACTCCTCCCC-3′; α5FRE/m3′, 5′-GATCAGCCGGGAGTTTGGCAAACTAAAAAAC-3′; α5FRE/m5′+3′, 5′-GATCAGCCGAAAAATTGGCAAACTAAAAAAC-3′), the DNA binding site for human HeLa CTF/NF-I in adenovirus type 2 (5′-GATCTTATTTTGGATTGAAGCCAATATGAG-3′) (37De Vries E. Van Driel W. Van den Heuvel S.J.L. Van der Vliet P.C. EMBO J. 1987; 6: 161-168Crossref PubMed Scopus (106) Google Scholar), the high affinity binding site for the positive transcription factor Sp1 (5′-GATCATATCTGCGGGGCGGGGCAGACACAG-3′) (38Dynan W.S. Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (911) Google Scholar), or the Sp1-binding site (designated p12.A) identified in the basal promoter from the mouse p12 gene (5′-GATCCAGTGGGTGGAGCCTG-3′) (36Robidoux S. Gosselin P. Harvey M. Leclerc S. Guérin S.L. Mol. Cell. Biol. 1992; 12: 3796-3806Crossref PubMed Scopus (37) Google Scholar). RCEC plated at either low (5 × 104 cells per 35-mm tissue culture plates), intermediate (5 × 105 cells per 35-mm tissue culture plates), or high (1, 5 × 106 cells per 35-mm tissue culture plates) cell density were transiently transfected using the polycationic detergent LipofectAMINE (Life Technologies, Inc.) as recommended by the manufacturer. Each LipofectAMINE-transfected plate received 1.5 μg of the test plasmid and 0.5 μg of the human growth hormone (hGH)-encoding plasmid pXGH5 (39Selden R.F. Burke-Howie K. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cel. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (471) Google Scholar). DrosophilaSchneider cells were transfected according to the calcium phosphate precipitation procedure (36Robidoux S. Gosselin P. Harvey M. Leclerc S. Guérin S.L. Mol. Cell. Biol. 1992; 12: 3796-3806Crossref PubMed Scopus (37) Google Scholar, 40Pothier F. Ouellet M. Julien J.-P. Guérin S.L. DNA Cell Biol. 1992; 11: 83-90Crossref PubMed Scopus (150) Google Scholar) at a density of 1 × 106 cells per 60-mm culture plate. Levels of CAT activity for all transfected cells were determined as described (40Pothier F. Ouellet M. Julien J.-P. Guérin S.L. DNA Cell Biol. 1992; 11: 83-90Crossref PubMed Scopus (150) Google Scholar) and normalized to the amount of hGH secreted into the culture media and assayed using a kit for quantitative measurement of hGH (Immunocorp, Montréal, Québec, Canada). Because the metallothionein-I promoter, which directs expression of hGH from the pXGH5 plasmid, proved to be highly inefficient inDrosophila cells, CAT activities from transfected Schneider cells were normalized to the amount of β-galactosidase encoded by the plasmid pAC5/V5-His/LacZ and cotransfected along with the CAT recombinant constructs. Each cell-containing plate therefore received 15 μg of the test plasmid, 4 μg of pAC5/V5-His/LacZ, and 1 μg of pPAC (empty vector). In the cotransfection experiments performed with the Sp1 expression plasmid, the empty pPAC was substituted for 1 μg of pPacSp1. The value presented for each individual test plasmid transfected corresponds to the mean of at least three separate transfections done in triplicate. To be considered significant, each individual value needed to be at least three times over the background level caused by the reaction buffer used (usually corresponding to 0.15% chloramphenicol conversion). Standard deviation is also provided for each transfected CAT plasmid. Crude nuclear extracts were prepared from RCEC grown solely on plastic or FN-coated culture dishes and dialyzed against DNase I buffer (50 mm KCl, 4 mm MgCl2, 20 mmK3PO4 (pH 7.4), 1 mmβ-mercaptoethanol, 20% glycerol) as described (41Roy R. Gosselin P. Guérin S.L. BioTechniques. 1991; 11: 770-777PubMed Google Scholar) except that a combination of protease inhibitors (pepstatin A (0.5 μg/ml), leupeptin (5 μg/ml), chymostatin (5 μg/ml), antipain (5 μg/ml), aprotinin (5 μg/ml), benzamidine (5 mm)) (all reagents from Sigma) was added to all the buffers used in order to restrict proteolysis. Extracts were kept frozen in small aliquots at −80 °C until use. EMSAs were carried out using either the 27-bp α5 FRE or the high affinity Sp1 oligomer as 5′ end-labeled probes. Approximately 2 × 104 cpm labeled DNA was incubated with crude nuclear proteins (as specified in the figure legends) from RCEC grown on either untreated or FN-coated culture dishes in the presence of 500 ng of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) in buffer D (5 mm HEPES (pH 7.9), 10% glycerol (v/v), 25 mm KCl, 0.05 mmEDTA, 0.5 mm dithiothreitol, 0.125 mmphenyl methosulfonyl fluoride). Occasionally, crude nuclear extracts from human HeLa cells were also used in EMSA as a positive control for comparison purposes. Incubation proceeded at room temperature for 10 min upon which time DNA-protein complexes were separated by gel electrophoresis through 6% native polyacrylamide gels run against Tris glycine buffer as described (42Schneider R. Dorper T. Gander I. Mertz R. Winnacker E.L. Nucleic Acids Res. 1986; 14: 1303-1317Crossref PubMed Scopus (94) Google Scholar). Gels were dried and autoradiographed at −80 °C to reveal the position of the shifted DNA-protein complexes generated. Competitions in EMSA were performed using 10 μg of crude nuclear proteins from RCEC grown in the presence of FN at 8 μg per cm2 as above except that molar excesses (100- and 500-fold) of synthetic double-stranded oligonucleotides bearing the DNA sequence of the α5 FRE, the DNA-binding site for human HeLa CTF/NF-I in adenovirus type 2 (37De Vries E. Van Driel W. Van den Heuvel S.J.L. Van der Vliet P.C. EMBO J. 1987; 6: 161-168Crossref PubMed Scopus (106) Google Scholar), the high affinity binding site for the positive transcription factor Sp1 (38Dynan W.S. Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (911) Google Scholar), or the p12.A Sp1-binding site from the mouse p12 gene (36Robidoux S. Gosselin P. Harvey M. Leclerc S. Guérin S.L. Mol. Cell. Biol. 1992; 12: 3796-3806Crossref PubMed Scopus (37) Google Scholar) were added to the binding reaction prior to loading on the gel. Supershift experiments in EMSA were conducted by first incubating varying amounts (as specified in the figure legends) of crude nuclear proteins from RCEC grown either with (8 μg per cm2) or without FN, in the presence of 250 ng of poly(dI-dC)·poly(dI-dC), with either none or 1 μl (corresponding to 1 μg) of a commercially engineered rabbit antiserum raised against the transcription factor Sp1 (Santa Cruz Biotechnology, Inc.) in buffer D. Then, 2 × 104 cpm FRE-labeled probe was added, and incubation was extended for another 15 min at room temperature. Samples were finally loaded on high ionic strength, 6% native polyacrylamide gels and run at 4 °C against Tris glycine buffer as above. Formation of DNA-protein complexes was revealed following autoradiography at −70 °C. Crude nuclear proteins were obtained from either HeLa cells (used as a positive control) or from RCEC grown on culture dishes coated or not with FN (8 μg per cm2) as detailed above. Protein concentration was evaluated by the Bradford procedure and further validated following Coomassie Blue staining of SDS-polyacrylamide fractionated nuclear proteins. One volume of sample buffer (6 m urea, 63 mm Tris (pH 6.8), 10% (v/v) glycerol, 1% SDS, 0,00125% (w/v) bromphenol blue, 300 mm β-mercaptoethanol) was added to 20 μg of proteins before they were size-fractionated on a 10% SDS-polyacrylamide minigel and transferred onto a nitrocellulose filter. A full set of protein molecular mass markers (Life Technologies, Inc.) was also loaded as a control to evaluate protein sizes. The blot was then washed once in TS buffer (150 mmNaCl, 10 mm Tris-HCl (pH 7.4)) and 4 times (5 min each at 22 °C) in TSM buffer (TS buffer plus 5% (w/v) fat free Carnation milk and 0.1% Tween 20). Then, a 1:500 dilution of a rabbit monoclonal antibody raised against the transcription factor Sp1 (Santa Cruz Biotechnology, Inc.) was added to the membrane-containing TSM buffer and incubation proceeded further for 4 h at 22 °C. The blot was then washed in TSM buffer and incubated an additional 1 h at 22 °C in a 1:1000 dilution of a peroxidase-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch). The membrane was successively washed in TSM (4 times, 5 min each) and TS (twice, 5 min each) buffers before immunoreactive complexes were revealed using Western blot chemiluminescence reagents (Renaissance, PerkinElmer Life Sciences) and autoradiographed. Studies conducted by Rajagopal et al. (23Rajagopal S. Huang S. Albitar M. Chakrabarty S. J. Cell. Physiol. 1997; 170: 138-144Crossref PubMed Scopus (14) Google Scholar) and Huang et al. (24Huang S. Varani J. Chakrabarty S. J. Cell. Physiol. 1994; 161: 470-482Crossref PubMed Scopus (19) Google Scholar) both provided evidence that the level of expression for the mRNA encoding the α5 integrin subunit was positively modulated by the presence of the extracellular matrix component fibronectin. We therefore exploited transient transfection of primary cultured RCEC using recombinant plasmids bearing the CAT reporter gene fused to various segments from the human α5 gene promoter (35Birkenmeier T.M. McQuillan J.J. Boedeker E.D. Argraves W.S. Ruoslahti E. Dean D.C. J. Biol. Chem. 1991; 266: 20544-20549Abstract Full Text PDF PubMed Google Scholar) in order to evaluate whether such an FN-dependent increase in α5 mRNA could be determined by discrete cis-acting elements from the α5 gene upstream regulatory region. For this purpose, a recombinant plasmid bearing the α5promoter up to position −954 (α5-954) inserted upstream from the CAT reporter gene was transfected into RCEC plated either on plastic or FN-coated culture dishes (2 μg/cm2) at varying cell densities. As Fig. 1 Aindicates, culturing RCEC on FN-coated Petris did not alter the activity driven by the α5−954 plasmid when transfected at low cell density (near 15% coverage of the plates). However, at both intermediate (near 75% coverage) and high (100% coverage for more than 48 h) cell density, the activity of the α5promoter was found to be 6.1- and 6.4-fold, respectively, higher when cells are grown on FN-coated culture plates rather than solely on plastic. Withdrawal of the serum contained into the culture medium (which normally contains 5% FBS) prior to cell seeding on FN-coated culture dishes had no statistical effect on the CAT activity directed by α5−954 (results not presented). The dose dependence of the α5 promoter FN responsiveness was next evaluated by transfecting RCEC plated at an intermediate cell density on culture dishes coated with either none or increasing concentrations of FN (from 1 to 16 μg/cm2). As shown on Fig. 1 B, the activity directed by the α5−954 plasmid increased proportionally to the amount of FN coated on the culture dishes, reaching a drastic 18-fold stimulation at 16 μg/cm2 FN. No further increase in α5promoter function was observed at FN concentrations above 16 μg/cm2 (results not presented). We therefore conclude that the activity of the human α5 promoter can be drastically increased when RCEC are grown on FN-coated culture dishes and that such a positive influence is obviously cell density-dependent. Discrete cis-acting regulatory elements are known to mediate many of the regulatory effects that are triggered through signal transduction pathways by binding trans-acting nuclear proteins with distinctive regulatory properties. To determine more precisely the minimal α5 promoter sequence required to confer FN responsiveness, CAT recombinant plasmids bearing various 5′ deletions of the α5 promoter were transfected into RCEC grown at intermediate density on both plastic and FN-coated (2 μg/cm2) culture dishes. Neither the deletion of the α5 promoter down to position −178 nor −92 could prevent the average 5-fold increase in α5 promoter activity observed when RCEC are grown on FN-coated plates. However, the further deletion of the α5 sequences down to position −41 almost totally abolished the FN responsiveness of the α5promoter. A detailed examination of this 41-bp sequence revealed the presence of a perfect inverted repeat of the following sequence, 5′-GGAGTTTG-3′ (Fig. 2 B). Therefore, FN responsiveness of the α5 promoter appears to be determined by a short stretch of DNA sequence contained between positions −41 and −92 relative to the α5 mRNA start site. Apart from FN, proteins such as collagen IV, vitronectin, entactin, and laminin are also commonly found in the extracellular matrix. We examined whether components from the ECM other than FN can also alter the expression directed by the α5 promoter in RCEC. For this purpose, RCEC were grown to intermediate cell density on either untreated or ECM-coated culture dishes before they were transiently transfected with the α5−954 plasmid. The ECM gel (basement membrane matrice from Engelbreth-Holm-Swarm mouse sarcoma, Fisher) contains laminin, collagen IV, entactin, and heparin sulfate proteoglycans but no FN. As shown on Fig. 3 A, the CAT activity driven by α5−954 is increased by only 2.5-fold when RCEC are grown on ECM-coated dishes (10 μg/cm2). The CAT activity directed by the α5 promoter was raised to 4-fold when both FN (2 μg/cm2) and the ECM gel (10 μg/cm2) are coated together on the culture dishes. However, optimal promoter activation was obtained when FN (2 μg/cm2) was coated alone on the culture plates (7.6-fold activation). Transient transfection of RCEC plated on ECM-coated culture dishes with the recombinant plasmids bearing the various 5′ deletions of the α5 promoter identified the ECM-responsive element somewhere between positions −178 and −954 (Fig. 3 B). We conclude that components from the ECM other than FN had only a moderate effect on the α5 promoter activity and that their action is mediated through a cis-acting element distinct from that which determines FN responsiveness in RCEC.Figure 7The α5 FRE can confer FN responsiveness to the basal promoter of the heterologous p12 gene. A, a synthetic oligonucleotide bearing the α5 inverted repeat from position −82 to −56 (α5 FRE) was inserted in either one (in plasmid p12/FRE) or two sense copies (in plasmid p12/2xFRE) upstream from the bas" @default.
• W2067982008 created "2016-06-24" @default.
• W2067982008 creator A5001010135 @default.
• W2067982008 creator A5062286994 @default.
• W2067982008 creator A5063844720 @default.
• W2067982008 creator A5078639626 @default.
• W2067982008 date "2000-12-01" @default.
• W2067982008 modified "2023-09-30" @default.
• W2067982008 title "Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro" @default.
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