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• W2023604509 abstract "We have demonstrated previously that a proximal element within the human α2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-γ (IFN-γ), and designated this region the IFN-γ response element (IgRE). Screening of a human fibroblast cDNA expression library with a radiolabeled IgRE probe exclusively yielded clones with a sequence identical to that of the transcription factor YB-1. Electrophoretic mobility shift assays (EMSA) using various IgRE-derived oligonucleotide probes containing serial two-base mutations showed that YB-1 protein was preferentially bound to the pyrimidine-rich sequence within the IgRE. This region is located immediately downstream of and partly overlaps the previously reported Sp1/Sp3 binding site. Overexpression of YB-1 in human dermal fibroblasts decreased steady state levels of COL1A2 mRNA and repressedCOL1A2 promoter activity in a dose-dependent manner. This inhibitory effect of YB-1 on COL1A2 expression was abolished by mutations of the IgRE shown to prevent YB-1 binding in EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-γ, suggesting that YB-1 mediates the inhibitory action of IFN-γ on COL1A2 promoter through its binding to the IgRE. Also, overexpression of a deletion mutant YB-1, which lacks the carboxyl-terminal domain, abrogated the repression ofCOL1A2 transcription by IFN-γ. A functional correlation between IFN-γ and YB-1 was further supported by luciferase assays using four tandem repeats of the Y-box consensus oligonucleotide linked to a minimal promoter. EMSA and Western blot analysis using cytoplasmic and nuclear proteins implied that IFN-γ promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-γ was further provided by using a YB-1-green fluorescent protein expression plasmid transfected into human fibroblasts. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-γ-elicited inhibition of COL1A2 expression, namely YB-1. We have demonstrated previously that a proximal element within the human α2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-γ (IFN-γ), and designated this region the IFN-γ response element (IgRE). Screening of a human fibroblast cDNA expression library with a radiolabeled IgRE probe exclusively yielded clones with a sequence identical to that of the transcription factor YB-1. Electrophoretic mobility shift assays (EMSA) using various IgRE-derived oligonucleotide probes containing serial two-base mutations showed that YB-1 protein was preferentially bound to the pyrimidine-rich sequence within the IgRE. This region is located immediately downstream of and partly overlaps the previously reported Sp1/Sp3 binding site. Overexpression of YB-1 in human dermal fibroblasts decreased steady state levels of COL1A2 mRNA and repressedCOL1A2 promoter activity in a dose-dependent manner. This inhibitory effect of YB-1 on COL1A2 expression was abolished by mutations of the IgRE shown to prevent YB-1 binding in EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-γ, suggesting that YB-1 mediates the inhibitory action of IFN-γ on COL1A2 promoter through its binding to the IgRE. Also, overexpression of a deletion mutant YB-1, which lacks the carboxyl-terminal domain, abrogated the repression ofCOL1A2 transcription by IFN-γ. A functional correlation between IFN-γ and YB-1 was further supported by luciferase assays using four tandem repeats of the Y-box consensus oligonucleotide linked to a minimal promoter. EMSA and Western blot analysis using cytoplasmic and nuclear proteins implied that IFN-γ promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-γ was further provided by using a YB-1-green fluorescent protein expression plasmid transfected into human fibroblasts. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-γ-elicited inhibition of COL1A2 expression, namely YB-1. Y-box-binding protein YB-1 mediates transcriptional repression of human α2(I) collagen gene expression by interferon-γ.Journal of Biological ChemistryVol. 278Issue 14PreviewPage 5160, Fig. 3 legend:Several symbols were incorrect. The symbols on lines 6 and 23 should becircles, not squares. Also, several words were incorrect. On lines 15–16, the parentheses should enclose the words 舠(open bar).舡 On lines 29–30, it should be 舠(closed bar).舡 On lines 39–40, it should be 舠(closed bar).舡 The corrected legend and the figure are shown below. Full-Text PDF Open Access Regulation of connective tissue formation is under rigorous control by cytokines, which act in concert to ensure tissue integrity during homeostasis, development and repair (1Mauviel A. Uitto J. Wounds. 1993; 5: 137-152Google Scholar). These cytokines have been shown to control connective tissue cell recruitment and proliferation, as well as synthesis and degradation of the extracellular matrix components (2Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar). Disruption of this equilibrium, leading to excessive collagen deposition, is the hallmark of interstitial fibrotic diseases. Type I collagen mRNA levels are increased in experimental and clinical fibrotic states (3Scharffetter K. Lankat-Buttgereit B. Kreig T. Eur. J. Clin. Invest. 1988; 18: 9-17Crossref PubMed Scopus (188) Google Scholar, 4Kulozik M. Hogg A. Lankat-Buttgereit B. Kreig T. J. Clin. Invest. 1990; 86: 917-924Crossref PubMed Scopus (212) Google Scholar), which implies a regulation at the transcriptional level. The role of transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; COL1A1 , α1(I) collagen gene; COL1A2 , α2(I) collagen gene; IFN-γ, interferon-γ; IgRE, IFN-γ response element; FCS, fetal calf serum; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assays; GFP, green fluorescence protein; CMV, cytomegalovirus; RT-PCR, reverse transcriptase PCR as the principal factor inducing collagen gene expression and leading to tissue fibrosis has been suggested by the observations that (a) TGF-β expression often parallels increased type I collagen gene expression (4Kulozik M. Hogg A. Lankat-Buttgereit B. Kreig T. J. Clin. Invest. 1990; 86: 917-924Crossref PubMed Scopus (212) Google Scholar, 5Peltonen J. Kahari L. Jaakkola S. Kahari V.M. Varga J. Uitto J. Jimenez S.A. J. Invest. Dermatol. 1990; 94: 365-371Abstract Full Text PDF PubMed Google Scholar), and (b) a soluble TGF-β receptor prevents collagen accumulation in experimental fibrosis (6Jacob G. Roulot D. Koteliansky V.E. Bissell D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12719-12724Crossref PubMed Scopus (322) Google Scholar). Several studies have previously indicated possible mechanisms by which tumor necrosis factor-α inhibits α2(I) collagen gene (COL1A2) expression at the transcriptional level (7Inagaki Y. Truter S. Tanaka S. DiLiberto M. Ramirez F. J. Biol. Chem. 1995; 270: 3353-3358Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 8Kouba D.J. Chung K.-Y. Nishiyama T. Vindevoghel L. Kon A. Klement J.F. Uitto J. Mauviel A. J. Immunol. 1999; 162: 4226-4234PubMed Google Scholar, 9Greenwel P. Tanaka S. Penkov D. Zhang W. Olive M. Moll J. Vinson C. Di Liberto M. Ramirez F. Mol. Cell. Biol. 2000; 20: 912-918Crossref PubMed Scopus (144) Google Scholar). Tumor necrosis factor-α has also been demonstrated to antagonize the effects of TGF-β through induction of inhibitory Smad7 (10Bitzer M. von Gersdorff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar) or AP-1 components (11Verrecchia F. Pessah M. Atfi A. Mauviel A. J. Biol. Chem. 2000; 275: 30226-30231Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). In contrast, identification of the transcription factors involved in interferon-γ-elicited down-regulation ofCOL1A2 expression has been elusive, except that a cross-talk between TGF-β/Smad and IFN-γ/Stat1 signaling pathways has recently been implicated in antagonistic regulation of gene transcription (12Ghosh A.K. Yuan W. Mori Y. Chen S.J. Varga J. J. Biol. Chem. 2001; 276: 11041-11048Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). We have previously identified a proximal element within the humanCOL1A2 promoter, spanning nucleotide −161 to −150, that mediates transcriptional repression by IFN-γ. We designated this region the IFN-γ response element (IgRE) (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar). UV cross-linking experiments using nuclear extracts prepared from IFN-γ-treated fibroblast cultures indicated that two DNA-protein complexes were formed with the IgRE (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar). Interestingly, others have shown that Sp1 and Sp3 bind to a TCCCCC motif located between −164 and −159 (14Ihn H. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1997; 272: 24666-24672Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), immediately upstream of and partly overlapping the IgRE. In the present study, we attempted to characterize the human fibroblast nuclear protein that interacts with the IgRE by screening of a human fibroblast cDNA expression library. We have identified the transcription factor YB-1 as a component of the COL1A2transrepressing complex bound to the IgRE. Cotransfection studies using a YB-1 expression vector and reporter constructs containing the wild-type and mutated COL1A2 IgRE have confirmed the role of YB-1 as a negative regulator of COL1A2 transcription mediating the effect of IFN-γ. A λgt 11 human dermal fibroblast cDNA library (Clontech) was screened by Southwestern blotting using the 32P-labeled three repeats of the IgRE oligonucleotide spanning −161 to −150 of the human COL1A2 promoter as described previously (15Vinson R.C. LaMarco L.K. Johnson F.P. Landschulz H.W. McKnight L.S. Genes Dev. 1988; 2: 801-806Crossref PubMed Scopus (346) Google Scholar). The filters were subjected to a cycle of denaturation with 6 mguanidine hydrochloride followed by renaturation before screening. After incubation with 5% skim milk in the binding buffer (10 mm Tris-HCl, pH 7.6, 50 mm NaCl, 10 mm MgC12, 1 mm EDTA, 1 mm dithiothreitol) for 30 min at 4 °C, the filters were probed in the binding buffer containing 106 cpm/ml probe, 10 μg/ml denatured salmon sperm DNA, and 0.25% powdered milk. The positive clones were sequenced directly using λgt 11 primers (Takara Biomedicals, Kyoto, Japan). Point mutations were introduced into the YB-1 binding sites using mutational polymerase chain reaction (PCR) as described previously (16Boast S. Su M.W. Ramirez M. Avvedimento E.V. J. Biol. Chem. 1990; 265: 13351-13356Abstract Full Text PDF PubMed Google Scholar, 17Chung K.-Y. Agarwal A. Uitto J. Mauviel A. J. Biol. Chem. 1996; 271: 3272-3278Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The constructs −161M1/luc and −161M5/luc containing two-base pair substitution mutations (5′-CCCATTCGCTCC-3′ to 5′-CGGATTCGCTCC-3′ and 5′-CCCATTCGCGGC-3′, respectively) were prepared by inserting the mutated promoter sequences into a firefly luciferase gene vector, pGL3 basic vector (Promega, Madison, WI). Four copies of the Y-box consensus oligonucleotide (CTGATTGGCTAA) linked to a minimal promoter containing only a TATA box were cloned into pGL3 basic vector. A YB-1 expression plasmid, YB-1/RSV, was constructed by ligating the entire YB-1 coding sequence into the HindIII/XbaI sites of pRc/RSV vector (Invitrogen). A deletion mutant YB-1 expression plasmid, which lacks the carboxyl terminus, was constructed by ligating the corresponding sequences into the BamHI/XhoI sites of pcDNA3.1(+) vector (Invitrogen). The pCMX-YB-1-GFP was prepared by ligating the YB-1 sequence into theHindIII/PmlI sites of pCMX-hGR-GFP (kindly provided by Dr. H. Ogawa, Kyoto University, Kyoto, Japan) (18Ogawa H. Umesono K. Acta Histochem. Cytochem. 1998; 31: 303-308Crossref Scopus (15) Google Scholar). The sequences of all plasmids were verified by automated sequencing (Applied Biosystems, Foster City, CA). Normal human dermal fibroblasts (Clontech, Palo Alto, CA) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS). Transient transfections were performed using the Lipofectin reagent (Invitrogen) according to the manufacturer's protocol. Forty hours after transfection, the cells were rinsed twice with phosphate-buffered saline, harvested by scraping, and lysed in lysis buffer (Promega). Aliquots containing the identical amounts of protein, as measured with a commercial assay kit (BioRad), were subjected to luciferase assays. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Fifty nanograms of total RNA was reverse-transcribed using ImProm-II reverse transcriptase (Promega). A pair of gene-specific PCR oligonucleotide primers and an oligonucleotide probe labeled with a reporter fluorescent dye at the 5′-end and a quencher fluorescent dye at the 3′-end were designed according to the guidelines suggested in the TaqMan model 5700 sequence detection instrument manual (Applied Biosystems). The human COL1A2 primers and probe used are as follows: forward primer, 5′-CCAGAGTGGAGCAGTGGTTACTACT-3′; reverse primer, 5′-TTCTTGGCTGGGATGTTTTCA-3′; and probe, 5′-CTACTGGCGAAACCTGTATCCGGGC-3′. The human YB-1 primers and probe used are as follows: forward primer, 5′-TCGCCAAAGACAGCCTAGAGA-3′; reverse primer, 5′-TCTGCGTCGGTAATTGAAGTTG-3′; and probe, 5′-TCAGCAGCCACCTCAACGTCGGTA-3′. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene primer and probe mixture from the predeveloped TaqMan assay reagents (Applied Biosystems) was used. The thermal cycling condition included 1 cycle at 50 °C for 5 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Standard curves for expression of each gene were generated by serial dilution of cDNA prepared from human fibroblasts. The relative mRNA expression levels ofCOL1A2 and YB-1 genes were normalized against those ofGAPDH gene in the same RNA preparation. Recombinant YB-1 protein was produced using the pET system (Novagen, Madison, WI) as described previously (19Takai T. Nishita Y. Ariga S. Ariga H. Nucleic Acids Res. 1994; 22: 5576-5581Crossref PubMed Scopus (34) Google Scholar). Briefly, after transformation of Escherichia coli BL21(DE3) with YB-1/pET-28a(+) expression vector, isopropyl-β-d-thiogalactopyranoside was added to LB medium at 0.5 A 600 followed by incubation for another 3 h. The induced cells were collected and sonicated until no longer viscous. The supernatant was applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen, Hilden, Germany), and recombinant (His)6-YB-1 was eluted with the buffer containing 200 mm imidazole. Purity of the expressed YB-1 fusion product was ascertained by analytic SDS polyacrylamide gel electrophoresis (PAGE). Nuclear and cytoplasmic extracts were prepared according to the method of Andrews and Faller (20Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). For EMSA, the probes (∼50,000 cpm) were incubated with recombinant YB-1 protein or nuclear extracts for 30 min on ice in 20 μl of binding reaction buffer as described previously (21Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (745) Google Scholar). For competition experiments, 20–500-fold molar excess of unlabeled IgRE or consensus sequences for YB-1 (22Stein U. Jurchott K. Walther W. Bergmann S. Schlag P.M. Royer H.D. J. Biol. Chem. 2001; 276: 28562-28569Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and Sp1/Sp3 (23Ihn H. Tamaki K. J. Invest. Dermatol. 2000; 114: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) were added to the binding reaction. In some experiments, antibody interference assays were performed by preincubating nuclear extracts with 2 μg of anti-YB-1 polyclonal antibodies prepared against the 15-amino acid peptide (residues 299–313) of YB-1 as previously reported (24Ohga T. Koike K. Ono M. Makino Y. Itagaki Y. Tanimoto M. Kuwano M. Kohno K. Cancer Res. 1996; 56: 4224-4228PubMed Google Scholar) or with anti-Sp1 and anti-Sp3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Twenty micrograms of nuclear proteins or 40 μg of cytoplasmic proteins were separated on a 7.5% SDS-polyacrylamide gel, electroblotted, and incubated with anti-YB-1 antibodies. ECL detection system (Amersham Biosciences) was used to detect immunoreactive proteins. Thirty-six hours after transfection with 1 μg of pCMV-YB-1-GFP using the LipofectAMINE Plus reagent (Invitrogen), 100 units/ml IFN-γ was added into the culture medium and incubated for another 6 h. After removing the media, cells were washed with phosphate-buffered saline and examined under a microscope (Nikkon, Tokyo, Japan) equipped with a fluorescein isothiocyanate filter set for fluorescence detection (18Ogawa H. Umesono K. Acta Histochem. Cytochem. 1998; 31: 303-308Crossref Scopus (15) Google Scholar). Values were expressed as mean ± S.D. Student's t test was used to evaluate the statistical differences between groups, and a p value of less than 0.05 was considered significant. A human dermal fibroblast cDNA library cloned into λgt 11 was screened using three tandem repeats of the IgRE (−161 to −150) as a probe. After screening ∼5 × 106 plaques, we obtained three positive clones. Direct sequence analyses of the three clones revealed overlapping subregions of the cDNA encoding YB-1, a member of the Y-box transcription factor family (data not shown). TheCOL1A2 IgRE sequence aligned in 7 of 12 bases with the consensus Y-box sequence (Fig. 1 A). Although YB-1 was identified originally as a transcription factor bound specifically to the Y-box sequence containing a CCAAT motif (25Didier D.K. Schiffenbauer J. Woulfe S.L. Zaceis M. Schwartz B.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7322-7326Crossref PubMed Scopus (358) Google Scholar), it has also been shown to bind to pyrimidine-rich oligonucleotides that can adopt an intramolecular triplex, single-stranded structure (26Kolluri R. Torrey A.T. Kinniburgh J.A. Nucleic Acids Res. 1992; 20: 111-116Crossref PubMed Scopus (121) Google Scholar, 27Wolffe A.P. BioEssays. 1994; 16: 245-251Crossref PubMed Scopus (327) Google Scholar). The IgRE within the human COL1A2 promoter possesses both the Y-box-like element and the pyrimidine-rich sequence (Fig. 1 A). To examine the binding specificity of YB-1 to the IgRE, a (His)6-YB-1 fusion protein was expressed in E. coli and used for gel mobility shift assays. Various double-stranded oligonucleotides containing serial two-base mutations or single-stranded oligonucleotides were used as probes to determine the recognition targets of YB-1 (Fig. 1 B). As shown in Fig. 1 C, recombinant YB-1 effectively bound to both the double-stranded (ds WT) and the sense single-stranded (ss-S WT) IgRE, whereas complex formation was not observed between YB-1 and the antisense single-stranded (ss-AS WT) IgRE. To further characterize DNA sequences involved in complex formation between YB-1 and double-stranded IgRE, a series of oligonucleotides containing substitution mutations were used as probes in EMSA. As shown in Fig. 1 C, M1 and M5 oligonucleotide probes completely failed to form the YB-1·DNA complexes, and M3 and M4 probes showed markedly diminished complex formation. In contrast, introduction of mutations into–158A−157T (M2) had no effect on the binding of YB-1 to the IgRE. These results suggest that both–160C−159C and–152T−151C bases were most essential for YB-1 binding to the IgRE. To determine whether endogenous YB-1 binds to the IgRE, we next performed EMSA using nuclear extracts prepared from human fibroblasts. As shown in Fig. 2 A, incubation of nuclear extracts with double-stranded IgRE probe yielded at least four retarded bands (arrows 1–4), all of which were diminished by the addition of increasing amounts of unlabeled double-stranded competitor. Interestingly, although formation of the three slowly migrating complexes (arrows 1–3) was completely abolished by adding a 20-fold molar excess of unlabeled double-stranded competitor, the faster migrating complex (arrow 4) was still observed even after adding a 500-fold molar excess of the competitor. These results suggested that the binding affinities of those nuclear proteins to the IgRE are different from each other. In addition, unlabeled sense single-stranded IgRE interfered with the formation of complex 4 in a dose-dependent manner (Fig. 2 A), whereas it did not affect complexes 1–3. On the other hand, antisense single-stranded IgRE as a competitor failed to diminish the formation of complex 4 (data not shown). These results clearly demonstrated that the nuclear factor(s) forming complex 4 preferentially bind to both the double- and the sense single-stranded IgRE. Considering the results shown in Fig. 1 C, we then tested the possibility that YB-1 interacts with the IgRE to form complex 4. For this purpose, we generated anti-YB-1 antibodies against the carboxyl terminus of YB-1, which inhibited the binding of recombinant YB-1 to the IgRE (Fig. 2 B). Preincubation of nuclear extracts with anti-YB-1 antibodies resulted in significant but partial inhibition of the complex 4 formation (Fig. 2 B). With regard to the slowly migrating complexes 1–3, a previous study indicated the binding of Sp1/Sp3 to the −164 to −159COL1A2 sequence, immediately upstream of and partly overlapping the IgRE (23Ihn H. Tamaki K. J. Invest. Dermatol. 2000; 114: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Consistent with their results, the formation of complex 1 was interfered with by anti-Sp1 antibodies and that of complexes 2 and 3 was abolished by adding anti-Sp3 antibodies (Fig. 2 B). Furthermore, in agreement with the results of Fig. 1 C, M1 and M5 oligonucleotides as a probe hardly formed the YB-1·IgRE complex (Fig. 2 C). Interestingly, interruption of YB-1 binding to the IgRE augmented the binding of Sp1 and Sp3 to the IgRE (Fig. 2 C). To confirm the transcriptional repression of COL1A2 expression by YB-1, we first performed real-time RT-PCR assays using Taqman probes. As shown in Fig. 3 A, expression of YB-1 suppressed endogenous COL1A2 mRNA levels in a dose-dependent manner. We then examined whether the binding of YB-1 to the IgRE is necessary for the regulation ofCOL1A2 promoter activity. To this end, we transfected human dermal fibroblasts with either the wild-type −161WT or with mutated −161M1 and −161M5 reporter plasmids together with a YB-1 expression plasmid. The basal transcription level of −161M1 was significantly higher than that of −161WT, whereas the basal transcription level of −161M5 was not statistically different from that of −161WT (Fig. 3 B). Overexpression of YB-1 significantly decreased transcription of −161WT construct by about 50% in human dermal fibroblasts. In contrast, the promoter activities of both the −161M1 and −161M5 constructs, which lack the YB-1 binding sequence within the IgRE, were not affected by overexpression of YB-1 (Fig. 3 B). Taken together, these results suggest that YB-1 is able to suppress COL1A2 gene expression and that the inhibitory action is exerted via a promoter region identified previously as the IgRE (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar). The results described above led us to investigate the role of YB-1 in mediating the inhibitory effect of IFN-γ onCOL1A2 promoter activity. We first performed real-time RT-PCR assays to confirm the transcriptional repression ofCOL1A2 by IFN-γ. As shown in Fig. 3 C, IFN-γ significantly suppressed endogenous COL1A2 mRNA levels in a dose-dependent manner. On the other hand, the levels of endogenous YB-1 mRNA remained unchanged, indicating that IFN-γ-elicited inhibition of COL1A2 transcription is independent of de novo synthesis of YB-1. Then, the mutated constructs −161M1 and −161M5, as well as their wild-type counterpart −161WT, were used in transient transfection experiments. Consistent with the results of our previous study (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar), IFN-γ exerted a significant transcriptional inhibition of the wild-type −161WT construct by about 50%. In contrast, the point mutations introduced into both –160C−159C nucleotides (–161M1) and–155T−154C nucleotides (–161M5) prevented the COL1A2 response to IFN-γ (Fig. 3 D). These results clearly demonstrate that the YB-1 binding site within the 12-base-pair IgRE is essential for IFN-γ responsiveness. To further confirm that YB-1 is involved inCOL1A2 repression by IFN-γ, we generated a deletion mutant YB-1 expression plasmid, which lacks the carboxyl-terminal region compared with the full-length YB-1 expression plasmid. As shown in Fig. 3 E, transcriptional repression of COL1A2by IFN-γ was strengthened further by overexpression of YB-1. On the other hand, overexpression of the mutant YB-1 abrogated theCOL1A2 repression by IFN-γ while keeping the basal transcription levels unchanged (Fig. 3 E). These results clearly demonstrated that YB-1 not only regulates the COL1A2transcription but also mediates IFN-γ elicited repression ofCOL1A2 transcription. To determine whether IFN-γ exerts its inhibitory effect solely through the YB-1 biding site or circumvents any additional non-YB-1-specific cis-elements, we constructed (YB-1)4-luciferase reporter plasmid in which four tandem repeats of the consensus Y-box sequence were linked to a minimal promoter containing only a TATA box. As shown in Fig. 4 A, expression of YB-1 decreased (YB-1)4-luciferase activity in a dose-dependent manner. Interestingly, a dose-dependent diminution of (YB-1)4-luciferase activity was also observed by changing the concentration of IFN-γ in culture medium (Fig. 4 B). These data suggest that IFN-γ exerts its inhibitory action specifically through the Y-box sequence. The results described above led us to investigate the IFN-γ-induced alterations in the relative amount and binding ability of YB-1 to the IgRE. We first evaluated alterations in the relative amount of YB-1 in response to IFN-γ using Western blot analysis. Twenty micrograms of nuclear proteins or 40 μg of cytoplasmic proteins prepared from cells either untreated or treated with IFN-γ for various lengths of time were coelectrophoresed with recombinant YB-1. The amount of YB-1 present in nuclear extracts was significantly increased by IFN-γ treatment for more than 4 h (Fig. 5 A,upper panel). Inversely, the amount of YB-1 present in cytoplasmic extracts was obviously decreased (Fig. 5 A,lower panel). Then we analyzed IFN-γ-induced alterations in the binding ability of YB-1 to the IgRE using EMSA. As shown in Fig. 5 B, the intensity of the YB-1·IgRE complex was increased remarkably by IFN-γ treatment. In contrast, the intensities of both the Sp1·IgRE and Sp3·IgRE complexes remained unchanged (data not shown). Furthermore, Southwestern blot analysis using IgRE as a probe showed that the intensity of the band estimated at ∼50 kDa, which is almost identical to the size of YB-1, was significantly increased by IFN-γ treatment for more than 4 h (data not shown). These results clearly demonstrated that IFN-γ promotes the nuclear translocation of YB-1 followed by its binding to the IgRE. To demonstrate directly the nuclear translocation of YB-1 by IFN-γ, human dermal fibroblasts were transfected with 1 μg of YB-1-GFP expression plasmid and treated with 100 units/ml IFN-γ. YB-1-GFP fusion protein was located mainly in the cytoplasm of untreated cells, whereas nuclear translocation of YB-1-GFP was observed as early as 4 h after exposure to IFN-γ (Fig. 6).Figure 6IFN-γ-induced nuclear translocation of YB-1. Human dermal fibroblasts were transiently transfected with 1 μg of pCMV-YB-1-GFP expression plasmid. Six hours after transfection, the cells were placed in medium supplemented with 10% FCS. After incubation for 40 h, the cells were left untreated (A) or treated (B) with 100 units/ml IFN-γ for 4 h. Then the cells were examined under a microscope equipped with a fluorescein isothiocyanate filter set for fluorescence detection.View Large Image Figure ViewerDownload (PPT) We have demonstrated previously that a proximal region of the human COL1A2 promoter is essential for mediating the inhibitory effect of IFN-γ and designated this region the IFN-γ response element, IgRE (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar). In the current study, by screening a human fibroblast cDNA expression library, we identified YB-1 as a transcription factor that interacts with the IgRE. Both bacterially expressed recombinant protein and endogenous YB-1 present in fibroblast nuclear extracts exhibited high affinity for the IgRE. Transient transfection assays demonstrated that expression of YB-1 decreasedCOL1A2 promoter activity in a dose-dependent manner and that IFN-γ suppressed gene transcription through YB-1 binding to the IgRE. Experiments using the carboxyl domain-deleted YB-1 confirmed the role of YB-1 in COL1A2 repression by IFN-γ. Furthermore, a combination of Western blot analysis, EMSA of nuclear proteins, and GFP fluorescence study demonstrated that IFN-γ treatment translocates YB-1 into the nucleus and increases the binding of YB-1 to the IgRE sequence. Ihn and co-workers (14Ihn H. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1997; 272: 24666-24672Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 23Ihn H. Tamaki K. J. Invest. Dermatol. 2000; 114: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) have demonstrated that ubiquitous transcription factors Sp1 and Sp3 bind to a TCCCCC motif located between −164 and −159 within the human COL1A2 promoter. Using transient transfection assays, they also showed that this pyrimidine-rich motif is a binding site for a transcriptional repressor (14Ihn H. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1997; 272: 24666-24672Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 23Ihn H. Tamaki K. J. Invest. Dermatol. 2000; 114: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 28Ihn H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1996; 271: 26717-26723Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, since the blockade of Sp1/Sp3 binding to the repressor site did not affect the collagen promoter activity (23Ihn H. Tamaki K. J. Invest. Dermatol. 2000; 114: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), the functional role of Sp1/Sp3 interacting with the TCCCCC motif has not been fully understood. Our present study has clearly indicated that the IgRE (−161 to −150), immediately downstream of and partly overlapping the Sp1/Sp3 binding site, is a binding site for a COL1A2repressor, YB-1. Indeed, Ihn et al. (28Ihn H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1996; 271: 26717-26723Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) introduced a substitution mutation into −161 CCC −159within the TCCCCC motif, which was almost comparable with our M1 mutation, and this resulted in a 6-fold increase in the basal promoter activity. Functional assays using four tandem repeats of the YB-1 consensus sequence indicated that IFN-γ exerts its inhibitory action onCOL1A2 transcription solely through the YB-1 binding site (Fig. 4). It should be noted, however, that the binding of Sp1/Sp3 was markedly enhanced when using the mutated IgRE sequences (M1 and M5) as EMSA probes (Fig. 2 C). YB-1 has been shown to interact with other transcription factors and viral proteins (29Safak M. Gallia G.L. Ansari S.A. Khalili K. J. Virol. 1999; 73: 10146-10157Crossref PubMed Google Scholar, 30Chernukhin I.V. Shaharum S. Robinson A.F. Carne A.F. Paul A. El Kady A.I. Lobanenkov V.V. Klenova E.M. J. Biol. Chem. 2000; 275: 29915-29921Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 31Okamoto T. Izumi H. Imamura T. Takano H. Ise T. Uchiumi T. Kuwano M. Kohno K. Oncogene. 2000; 19: 6194-6202Crossref PubMed Scopus (134) Google Scholar). In addition, a previous study has suggested that the level of Sp1 activity dictates binding of YB-1 to its target sequence and therefore affects its regulatory function (32Sawaya B.E. Khalili K. Amini S. J. Gen. Virol. 1998; 79: 239-246Crossref PubMed Scopus (29) Google Scholar). Thus, it could be argued that YB-1 and Sp1/Sp3 regulate COL1A2 transcription through their physical interactions and/or competitive binding to the adjacent IgRE and the TCCCCC motif. In contrast to the mutation introduced into–160C−159C, the other substitution mutation introduced into another essential YB-1 binding site (–155T−154C) did not affect the basal promoter activity (Fig. 3). We suggested previously (13Higashi K. Kouba D.J. Song Y.-J. Uitto J. Mauviel A. Matrix Biol. 1998; 16: 447-456Crossref PubMed Scopus (39) Google Scholar) that a protein with size estimated at ∼30 kDa definitely interacts with the IgRE through the –155T−154C nucleotides and acts as a transactivator. It is therefore possible that the lack of binding of both YB-1 and the 30-kDa protein did not affect promoter activity. In addition, Southwestern blot analyses showed that IFN-γ treatment increased YB-1 binding to the IgRE, whereas it had no effect on the binding of the 30-kDa protein (data not shown). Altogether, reciprocal interactions between YB-1, Sp1/Sp3, and the 30-kDa protein might be required for regulation of COL1A2 transcription by IFN-γ. The significant but partial inhibition of YB-1·IgRE complex formation by adding anti-YB-1 antibodies (Fig. 2 B) may indicate that the 30-kDa protein interacts with YB-1 and interfere with its antibody recognition. The nature of the 30-kDa protein as well as the mechanisms of its functional interaction with YB-1 also remain to be elucidated. It has been shown that chk-YB-1b, a chicken homologue of human YB-1, binds to a pyrimidine-rich sequence and activates the rat α1(I) procollagen gene (COL1A1) transcription (33Dhalla A.K. Ririe S.S. Swamynathan S.K. Weber K.T. Guntaka R.V. Biochem. J. 1998; 336: 373-379Crossref PubMed Scopus (20) Google Scholar). Amino acid sequences of the nucleic acid binding domain and the carboxyl terminus of chk-YB-1b display a high degree of homology with those of human YB-1. In contrast, the sequence of the amino-terminal domain, which may influence the ability of the protein to interact with other nucleic acid binding proteins, is the most structurally distinct portion (34Grant C.E. Deeley R.G. Mol. Cell. Biol. 1993; 13: 4186-4196Crossref PubMed Scopus (83) Google Scholar). Norman et al. (35Norman J.T. Lindahl G.E. Shakib K. En Nia A. Yilmaz E. Mertens P.R. J. Biol. Chem. 2001; 276: 29880-29890Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) have shown that YB-1 overexpression suppresses endogenous COL1A1 expression and collagen protein production in rodent cells. Sequence analysis of the mouseCOL1A1 promoter revealed three putative YB-1 binding sites, −83/–72, −103/–92, and −129/–118, all of which are well conserved in human (35Norman J.T. Lindahl G.E. Shakib K. En Nia A. Yilmaz E. Mertens P.R. J. Biol. Chem. 2001; 276: 29880-29890Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, Yuan et al. (36Yuan W. Yufit T. Li L. Mori Y. Chen S.-J. Varga J. J. Cell. Physiol. 1999; 179: 97-108Crossref PubMed Scopus (55) Google Scholar) have demonstrated transcriptional inhibition of human COL1A1expression by IFN-γ and located the IFN-γ response element between nucleotides −129 and −109 of the human COL1A1 promoter. In the present study, we have shown that YB-1 suppresses transcription of human COL1A2 gene and that IFN-γ inhibits gene transcription via the YB-1 binding site. Taken together, these results suggest that IFN-γ/YB-1 signaling may coordinately down-regulate bothCOL1A1 and COL1A2 gene transcription. Consensus sequence for translocation of proteins to the nucleus does not exist in YB-1. However, nuclear translocation signals are located in its carboxyl terminus, which typically contain a cluster of three to six basic residues in a short peptide of four to nine amino acids (25Didier D.K. Schiffenbauer J. Woulfe S.L. Zaceis M. Schwartz B.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7322-7326Crossref PubMed Scopus (358) Google Scholar). In this study, deletion of the carboxyl-terminal domain of YB-1 containing those nuclear translocation signals resulted in an abrogation of COL1A2 repression by IFN-γ. Recently, Stenina et al. (37Stenina O.I. Shaneyfelt K.M. DiCorleto P.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7277-7282Crossref PubMed Scopus (47) Google Scholar) have suggested that thrombin induces the release of YB-1 from mRNA by proteolytic cleavage and the truncated YB-1 is translocated into the nucleus and bound to the thrombin response element (CCACCCACC) in endothelial cells. However, Western blot analyses using an amino-terminally Flag-tagged YB-1 expression vector failed to show degradation of YB-1 following IFN-γ treatment of human fibroblasts (data not shown). In contrast, a previous study using human cancer cells showed that the nuclear translocation of YB-1 is induced by UV irradiation or anticancer drugs like cisplatin in a protein kinase C-dependent manner (38Koike K. Uchiumi T. Ohga T. Toh S. Wada M. Kohno K. Kuwano M. FEBS Lett. 1997; 417: 390-394Crossref PubMed Scopus (176) Google Scholar). Our results showed that treatment with 100 nm12-O-tetradecanoylphorbol-13-acetate also initiated the nuclear translocation of YB-1 in human fibroblasts (data not shown). These results may suggest that some of the IFN-γ-induced biological events, such as the induction of YB-1 translocation or the inhibition of type I collagen gene expression, are mediated through the protein kinase C pathway (39Reano A. Viac J. Richard M.H. Schmitt D. Arch. Dermatol. Res. 1997; 289: 617-622Crossref PubMed Scopus (2) Google Scholar, 40Lin Y.H. Martino J.L. Wilcox D.B. Davis B.F. Gordinier K.J. Davis J.P. J. Immunol. 1998; 161: 843-849PubMed Google Scholar). The precise mechanism by which IFN-γ initiates the nuclear translocation of YB-1 is currently under investigation. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-γ-elicited inhibition ofCOL1A2 expression, namely YB-1. Future studies aimed at the characterization of the molecular mechanism involved in the regulation of type I collagen gene expression by YB-1 may provide a novel insight into the interstitial fibrotic diseases and eventually contribute to the development of useful therapeutic means. We thank Dr. Hidesato Ogawa in Kyoto University for the generous gift of pCMX-hGR-GFP plasmid. We also thank Dr. Ko Fujimori for valuable comments on this study and Yukiko Miyama for excellent technical assistance." @default.
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