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• W2022894345 abstract "Previously we determined that inflammation responsive transcription factors AP-1 and SAF-1 synergistically regulate transcriptional induction of the MMP-1 gene. The present study investigated the underlying molecular mechanism of cooperativity between these two different groups of transcription factors. We present evidence that knockdown of SAF-1 by small interfering RNAs inhibits AP-1-mediated increase of human MMP-1 expression. The two key members of the AP-1 family of proteins, c-Fos and c-Jun, and SAF-1 form a ternary protein complex, which has markedly higher DNA binding activity than either a SAF-1 homodimer or a c-Fos/c-Jun heterodimer. The increased DNA binding activity of the ternary complex is translated into a striking enhancement of their transcriptional activity by which synergistic transcriptional induction of MMP-1 expression is achieved. The SAF-1·c-Fos·c-Jun ternary complex efficiently promotes transcription from both SAF-1 and AP-1 sites of human MMP-1 promoter. The physical interaction between SAF-1 and AP-1 was demonstrated both in vitro by Far-Western and antibody pulldown assays with recombinant proteins and in vivo by chromatin immunoprecipitation (ChIP), re-ChIP, and co-immunoprecipitation analyses. Two distinct but adjacent domains in SAF-1 are involved in protein-protein contact with c-Fos and c-Jun; one domain resides within two N-terminal polyalanine tracts, and the other is present within the first two zinc finger motifs. Together these findings delineate the mechanism of synergy and the essential role of SAF-1 and AP-1 in up-regulating human MMP-1 expression under various inflammatory conditions. Previously we determined that inflammation responsive transcription factors AP-1 and SAF-1 synergistically regulate transcriptional induction of the MMP-1 gene. The present study investigated the underlying molecular mechanism of cooperativity between these two different groups of transcription factors. We present evidence that knockdown of SAF-1 by small interfering RNAs inhibits AP-1-mediated increase of human MMP-1 expression. The two key members of the AP-1 family of proteins, c-Fos and c-Jun, and SAF-1 form a ternary protein complex, which has markedly higher DNA binding activity than either a SAF-1 homodimer or a c-Fos/c-Jun heterodimer. The increased DNA binding activity of the ternary complex is translated into a striking enhancement of their transcriptional activity by which synergistic transcriptional induction of MMP-1 expression is achieved. The SAF-1·c-Fos·c-Jun ternary complex efficiently promotes transcription from both SAF-1 and AP-1 sites of human MMP-1 promoter. The physical interaction between SAF-1 and AP-1 was demonstrated both in vitro by Far-Western and antibody pulldown assays with recombinant proteins and in vivo by chromatin immunoprecipitation (ChIP), re-ChIP, and co-immunoprecipitation analyses. Two distinct but adjacent domains in SAF-1 are involved in protein-protein contact with c-Fos and c-Jun; one domain resides within two N-terminal polyalanine tracts, and the other is present within the first two zinc finger motifs. Together these findings delineate the mechanism of synergy and the essential role of SAF-1 and AP-1 in up-regulating human MMP-1 expression under various inflammatory conditions. A crucial role of matrix metalloproteinase-1 (MMP-1) 3The abbreviations used are: MMP, matrix metalloproteinase; AP-1, activator protein 1; ECM, extracellular matrix; SAF, serum amyloid A-activating factor; MAP, mitogen-activated protein; siRNA, small interfering RNA; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; RT, reverse transcription; IL, interleukin.3The abbreviations used are: MMP, matrix metalloproteinase; AP-1, activator protein 1; ECM, extracellular matrix; SAF, serum amyloid A-activating factor; MAP, mitogen-activated protein; siRNA, small interfering RNA; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; RT, reverse transcription; IL, interleukin. in the pathogenesis of various diseases associated with extracellular matrix (ECM) degradation is well established (1Ehrlich M.G. J. Orthop. Res. 1985; 3: 170-184Crossref PubMed Scopus (24) Google Scholar, 2Berend K.R. Toth A.P. Harrelson J.M. Layfield L.J. Hey L.A. Scully S.P. J. Bone Jt. Surg. Am. 1998; 80: 11-17Crossref PubMed Scopus (38) Google Scholar, 3Galis Z.S. Sukhova G.K. Kranzhofer R. Clark S. Libby P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 402-406Crossref PubMed Scopus (513) Google Scholar). MMP-1 is a prominent member of more than 26 structurally related matrix MMP proteins that in concert are capable of degrading all components of the ECM at physiological pH (4Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3592) Google Scholar, 5Birkedal-Hansen H. Curr. Opin. Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (974) Google Scholar). Although all MMPs possess some ECM-degrading capacity, the initial cleavage of triple helical collagen can be done only by a small subset of this family of which MMP-1 is one. Normal expression of MMP-1 is thus at a very low level and is highly regulated. Under many pathogenic conditions, MMP-1 protein concentration is markedly increased, primarily by transcriptional induction of the MMP-1 gene. Increase of MMP-1 mRNA by many inflammatory stimuli such as cytokines, growth factors, and tumor promoting agents is still a major challenge in successful inhibition of this molecule for the treatment of all MMP-1-related pathogenicities (6Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar).Several transcription factors have been implicated thus far for transcriptional induction from the MMP-1 gene; these include activator protein 1 (AP-1) (7Angel P. Baumann I. Stein B. Delius H. Rahmsdorf H.J. Herrlich P. Mol. Cell. Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (583) Google Scholar) and serum amyloid A-activating factor 1 (SAF-1) (8Ray A. Kuroki K. Cook J.L. Bal B.S. Kenter K. Aust G. Ray B.K. Arthritis Rheum. 2003; 48: 134-145Crossref PubMed Scopus (31) Google Scholar). AP-1 comprises a group of proteins containing a basic leucine zipper DNA-binding domain. AP-1 family members consist of the Fos (c-Fos, Fra-1, Fra-2, FosB) and Jun (c-Jun, JunB, and JunD) proteins (9Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131Crossref PubMed Scopus (2164) Google Scholar, 10Ransone L.J. Verma I.M. Annu. Rev. Cell Biol. 1990; 6: 539-557Crossref PubMed Scopus (343) Google Scholar). Typically, AP-1 protein dimers bind to AP-1-binding elements, and two subunits are formed either by heterodimerization or homodimerization. In cells, the Fos/Jun heterodimer is the most predominant form of AP-1, which has a very high affinity for an AP-1 DNA-binding element, whereas the Jun/Jun homodimer exhibits a very low affinity for the same DNA. AP-1 proteins are transcriptionally induced by the action of various serum and growth factor regulated factors. For example, the c-fos gene is transcriptionally induced by the mitogen-activated protein (MAP) kinase-activated Elk transcription factor, a member of the ternary complex factor (TCF) family, (11Hipskind R.A. Rao V.N. Mueller C.G. Reddy E.S. Nordheim A. Nature. 1991; 354: 531-534Crossref PubMed Scopus (350) Google Scholar, 12Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar) and by protein kinase C during oncogenic Ha-Ras-mediated induction (13Kampfer S. Hellbert K. Villunger A. Doppler W. Baier G. Grunicke H.H. Uberall F. EMBO J. 1998; 17: 4046-4055Crossref PubMed Scopus (62) Google Scholar). SAF-1 transcription factor is a member of the multiple Cys2-His2-type zinc finger proteins, which also are activated in response to various inflammatory stimuli, including cytokines, phorbol 12-myristate 13-acetate, lipopolysaccharide, and oxidized low density lipoproteins (14Ray A. Ray B.K. Mol. Cell. Biol. 1998; 18: 7327-7335Crossref PubMed Scopus (42) Google Scholar, 15Ray A. Schatten H. Ray B.K. J. Biol. Chem. 1999; 274: 4300-4308Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16Ray B.K. Chatterjee S. Ray A. DNA Cell Biol. 1999; 18: 65-73Crossref PubMed Scopus (26) Google Scholar, 17Ray B.K. Ray A. Biochemistry. 1997; 36: 4662-4668Crossref PubMed Scopus (34) Google Scholar). Phosphorylation of SAF-1 via MAP kinase, protein kinase C, or protein kinase A markedly increases its DNA binding and transactivation potential (18Ray A. Yu G.Y. Ray B.K. Mol. Cell. Biol. 2002; 22: 1027-1035Crossref PubMed Scopus (33) Google Scholar, 19Ray A. Fields A.P. Ray B.K. J. Biol. Chem. 2000; 275: 39727-39733Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 20Ray A. Ray P. Guthrie N. Shakya A. Kumar D. Ray B.K. J. Biol. Chem. 2003; 278: 22586-22595Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar).The activation of AP-1 and SAF-1 by similar signal transduction cascades, the MAP kinase and protein kinase C pathways, suggests that these transcription factors may function in a cooperative manner and/or may be dependent on each other to exert their full transactivation potential. A recent study from our laboratory indicates that concurrent participation of AP-1 and SAF-1 is necessary for full transcriptional induction of canine MMP-1 (21Ray A. Shakya A. Ray B.K. Biochim. Biophys. Acta. 2005; 1732: 53-61Crossref PubMed Scopus (18) Google Scholar). The SAF-1 and AP-1 DNA-binding elements are well conserved in the MMP-1 promoter among many species, which further underscore their importance. Functional cooperation between candidate proteins, in general, is mediated by protein-protein interaction, which is most effective when the DNA-binding elements of the involved transcription factors are proximally located. In correlation with this hypothesis, several studies are available that demonstrate the ability of nuclear proteins to bind each other and to mediate cooperative DNA binding and promoter activation when their respective binding elements are juxtaposed (22Gutman A. Wasylyk B. Trend. Genet. 1991; 7: 49-54Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 23Fry C.J. Farnham P.J. J. Biol. Chem. 1999; 274: 29583-29586Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). However, in human or canine MMP-1, SAF-1 and AP-1 DNA-binding elements are not in close proximity but are more than 250 nucleotides apart. To understand the mechanism of cooperation between SAF-1 and AP-1, we have extended the study of human MMP-1, and in this report we present evidence for a physical interaction between these proteins, the formation of a ternary protein complex with markedly high DNA binding ability and a synergistic level of transactivation potential. Further, this study provides, for the first time, an identification of novel structural motifs in the SAF-1 transcription factor for protein-protein contact with AP-1 family members. These findings extend the paradigm that cellular machinery utilizes concurrent activation of a network of transcription factors and their cooperative interaction for efficient and optimal activation of genes.EXPERIMENTAL PROCEDURESCell Culture and Transfection—HTB-94 human chondrocyte cells derived from a primary grade II chondrosarcoma were cultured in Dulbecco's modified Eagle's medium containing high glucose, 100 units/ml penicillin, and 100 units/ml streptomycin supplemented with 7% fetal calf serum. Transfection assays were performed as described previously (8Ray A. Kuroki K. Cook J.L. Bal B.S. Kenter K. Aust G. Ray B.K. Arthritis Rheum. 2003; 48: 134-145Crossref PubMed Scopus (31) Google Scholar). The pSV-β galactosidase (Promega) plasmid DNA was used as an internal control and was assayed as described (24Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 2001; (Ed. 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)Google Scholar). Cells were harvested 24 h post-transfection, and CAT activity was measured as described previously (24Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 2001; (Ed. 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)Google Scholar). All transfection experiments were performed at least three times.Plasmids—MMP-1(-518/+63)-CAT reporter was constructed by ligating the human MMP-1 genomic DNA sequence from nucleotide position -518 to +63 into pBLCAT3 vector (25Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). Mutant MMP-1-CAT reporters were constructed by ligating the respective mutant DNA fragments generated by megaprimer PCR into pBLCAT3 vector. Mutant SAF-1 and AP-1 oligonucleotide sequences used were 5′-CCTTGCTGACACTGCAGAATGTGGAATTACTAAC-3′ and 5′-GGATGTTATAAAGCAGATCTTAGACAGCC-3′, respectively. The underlined bases represent altered sequences. The SAF-1 CAT (26Ray A. Ray B.K. Mol. Cell. Biol. 1996; 16: 1584-1594Crossref PubMed Scopus (40) Google Scholar) or AP-1 CAT reporter plasmids contained three tandem copies of SAF-1 or AP-1 DNA-binding elements into pBLCAT2 vector. Various SAF-1 fragments were constructed by PCR, and wherever it became necessary, initiator ATG and TGA stop codons were added to the coding sequences. The PCR-amplified DNAs were ligated to the appropriate expression vectors, including pcDNA3 (Invitrogen), pGEX-5X-1 (GE Healthcare), PRSET (Invitrogen), and pAR (27Blanar M.A. Rutter W.J. Science. 1992; 256: 1014-1018Crossref PubMed Scopus (273) Google Scholar). Primer sets used for synthesizing various SAF-1 derivatives, indicated by beginning and ending amino acids in parentheses, were as follows: SAF-1-(1-477), 5′-ATGTTCCCCGTGTTCCCTTGCACGCTG-3′ and 5′-AGCTCACCAAGGCTGGGAGGGAAGTGG-3′; SAF-1-(1-90), 5′-ATGTTCCCCGTGTTCCCTTGCACGCTG-3′ and 5′-TCAGAGAACCGGGAGCAAGTCCAC-3′; SAF-1-(1-192), 5′-ATGTTCCCCGTGTTCCCTTGCACGCTG-3′ and 5′-TCAGGGCCCCTTGCTCTTTGTCTTCTTC-3′; SAF-1-(90-192), 5′-ATGCCGGTTCTCGCCGCCGCGCAGGAATC-3′ and 5′-TCAGGGCCCCTTGCTCTTTGTCTTCTTC-3′; SAF-1-(192-360), 5′-ATGAAGGGGCCCTACATTTGCGCCCTG-3′ and 5′-TCACTCTGTTGAGTGCACTTGTCTGAC-3′; SAF-1-(192-413), 5′-ATGAAGGGGCCCTACATTTGCGCCCTG-3′ and 5′-TCACTGGCTGTGCACCTTCATGTGG-3′; SAF-1-(90-301), 5′-ATGCCGGTTCTCGCCGCCGCGCAGGAATC-3′ and 5′-TCAGTCCGAGTGGGAGAGCTTGTGTCG-3′; SAF-1-(192-301), 5′-ATGAAGGGGCCCTACATTTGCGCCCTG-3′ and 5′-TCAGTCCGAGTGGGAGAGCTTGTGTCG-3′; SAF-1-(413-477), 5′-ATGGCCAGGGTCCTCACCATGTCTGTG-3′ and 5′-AGCTCACCAAGGCTGGGAGGGAAGTGG-3′.Protein Preparation—Bacterially expressed proteins were purified by affinity chromatography using nickel-agarose (Invitrogen) or glutathione-Sepharose column chromatography following the manufacturer's protocol. pET-cJun and pET-cFos plasmid DNAs were obtained from James A. Goodrich, and the c-Jun/c-Fos heterodimer was prepared as described (28Ferguson H.A. Goodrich J.A. Nucleic Acids Res. 2001; 29: E98Crossref PubMed Scopus (17) Google Scholar).GST Pulldown Assay—GST-tagged full-length SAF-1 or truncated SAF-1 proteins and control GST proteins (50 pmol each) were mixed individually with glutathione-Sepharose 4B beads (Amersham Biosciences), incubated for 1 h at 4 °C in binding buffer (12 mm HEPES, pH 7.9, 4 mm Tris-HCl, pH 7.9, 100 mm NaCl, 1 mm EDTA), and washed extensively (five times) with binding buffer. Next, GST-SAF-1, truncated GST-SAF-1, or control GST protein immobilized to Sepharose beads was mixed with bacterially expressed c-Jun/c-Fos heterodimer, incubated for 1 h at 4 °C, and washed extensively with binding buffer plus 0.05% Nonidet P-40. The proteins were eluted using elution buffer (50 mm Tris-HCl, pH 8.0, 100 mm reduced glutathione) or boiled in protein sample loading buffer containing 1% SDS plus β-mercaptoethanol, analyzed by SDS-PAGE using 11% polyacrylamide gel, and visualized by Coomassie Blue staining.Far-Western Assay—Equal amounts of protein were electrophoresed on 11% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated in a modified HEPES binding buffer (25 mm HEPES, pH 7.9, 25 mm NaCl, 5 mm MgCl2, 5 mm β-mercaptoethanol) containing 6 m guanidine hydrochloride for 10 min at room temperature. Membrane-bound denatured proteins were renatured by sequential incubation of the membrane for five cycles (10 min each) in buffers that were diluted each time (1:1) with HEPES binding buffer without any guanidine hydrochloride. The membrane received a final rinse twice in HEPES binding buffer and then were incubated in a blocking solution (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 5% bovine serum albumin) at 4 °C overnight, further incubated in overlay buffer (0.5% bovine serum albumin, 0.25% gelatin, 1% Nonidet P-40, 10 mm NaCl, 1 mm EDTA, 5 mm β-mercaptoethanol, 20 mm HEPES, pH 7.5) containing 32P-labeled pAR-SAF-1 at room temperature for 4 h, washed four times for 10 min each in a washing buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.5% sarcosyl), dried, and autoradiographed. The pAR-SAF-1 protein was prepared by cloning a full-length SAF-1 cDNA in pAR vector (27Blanar M.A. Rutter W.J. Science. 1992; 256: 1014-1018Crossref PubMed Scopus (273) Google Scholar) that contains a FLAG tag and a heart muscle kinase phosphorylation site, and purified protein was radioactively labeled with [γ-32P]ATP and heart muscle kinase (Sigma).siRNA Experiments—HTB-94 cells were transfected in duplicate with predesigned human MAZ/SAF-1-specific siRNAs (Dharmacon Research, Lafayette, CO) using Oligofectamine (Invitrogen) according to the manufacturer's protocol. As negative controls, mock transfection (no siRNA) and nonspecific scrambled siRNAs were used. Forty-eight hours after transfection, cells in each treatment groups were harvested and divided into two parts. Also conditioned media were collected for measuring secreted MMP-1 and MMP-2 proteins. Total RNA was isolated from one portion of the cells using the guanidinium thiocyanate method (29Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar), and RT-PCR was performed using an RT-PCR kit from Invitrogen. The PCR primer sequences specific for the genes examined and the predicted product sizes, shown in parentheses, were: MAZ/SAF-1 (205 bp), 5′-GCCAGGGTCCTCACCATGTCTG-3′ (sense) and 5′-CAACTTGGAGCTCACCAGGG-3′ (antisense); MMP-1 (234 bp), 5′-ATGCTGAAACCCTGAAGGTG-3′ (sense) and 5′-CTGCTTGACCCTCAGAGACC-3′ (antisense); and GAPDH (178 bp) 5′-TGCACCACCAACTGCTTAG-3′ (sense) and 5′-TAGAGGCAGGGATGATGTTC-3′ (antisense). The second set of cells was used for Western blot analysis using either anti-SAF-1 (prepared as described previously (8Ray A. Kuroki K. Cook J.L. Bal B.S. Kenter K. Aust G. Ray B.K. Arthritis Rheum. 2003; 48: 134-145Crossref PubMed Scopus (31) Google Scholar)). The conditioned media were concentrated by freeze-drying in a lyophilizer, and the protein contents were measured. Fifty micrograms of proteins were subjected to Western blotting using anti-MMP-1 or anti-MMP-2 antibody (Santa Cruz Biotechnology).For cotransfection analysis, siRNAs were transfected into HTB-94 cells following the manufacturer's protocol, and 24 h after transfection the medium was replaced and further transfected with MMP-1-CAT reporter plasmid and pcDc-Fos plus pcDc-Jun plasmids. Cells were harvested 24 h later and used for the CAT assay.Electrophoretic Mobility Shift Assay—Equal amounts (0.1 μg) of bacterially expressed proteins were used for an electrophoretic mobility shift assay, which was performed as described previously (17Ray B.K. Ray A. Biochemistry. 1997; 36: 4662-4668Crossref PubMed Scopus (34) Google Scholar). Protein concentrations were measured by the Bradford method (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar). Radiolabeled probes were prepared by labeling double-stranded oligonucleotides with [α-32P]dCTP. In some assays, anti-SAF-1 or anti-c-Fos and anti-c-Jun (Santa Cruz Biotechnology) antibodies were added during a 30-min preincubation on ice prior to the addition of the labeled probe. Anti-SAF-1 antibody was prepared as described (8Ray A. Kuroki K. Cook J.L. Bal B.S. Kenter K. Aust G. Ray B.K. Arthritis Rheum. 2003; 48: 134-145Crossref PubMed Scopus (31) Google Scholar). In some assays, 25 pmol of the following oligonucleotides were added in the preincubation mixtures as competitor oligonucleotides: SAF-1 oligo, 5′-CCCTTCCTCTCCACCCACAGCCCCCATGG-3′; AP-1 oligo, 5′-CGCTTGATGAGTCAGCCGGAA-3′; and nonspecific oligo, 5′-TGTCGAATGCAAATCACTAGAA-3′.Chromatin Immunoprecipitation (ChIP) and Re-ChIP Assays—ChIP was performed with a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY) following the manufacturer's protocol. Briefly, HTB-94 cells (2 × 106 each) were stimulated with IL-1β (500 units/ml). Twenty-four hours later, formaldehyde (1% final concentration) was added directly to the culture medium and incubated at 37 °C for 10 min for cross-linking. Cells were lysed in 200 μl of cell lysis buffer (50 mm Tris-HCl, pH 8.0, 10 mm EDTA, 1% SDS) with a protease inhibitor mixture and sonicated to generate ∼500-bp long DNA fragments, and the supernatants were diluted with a dilution buffer (20 mm Tris-HCl, pH 8.0, 1.0 mm EDTA, 150 mm NaCl, 1% Triton X-100, 0.01% SDS, and protease inhibitors). The solutions were precleared with a salmon sperm DNA/protein G-agarose slurry and then incubated with anti-SAF-1 antibody or a mixture of anti-c-Fos and anti-c-Jun antibodies (Santa Cruz Biotechnology) at 4 °C for 16 h with rotation. The immune complexes were precipitated with salmon sperm DNA/protein G-agarose. The agarose beads were washed sequentially in a low salt wash buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton-X-100, and 0.1% SDS), a high salt wash buffer (same as low salt wash buffer except containing 500 mm NaCl), LiCl wash buffer (20 mm Tris-HCl, pH 8.0, 250 mm LiCl, 0.5% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors), and 20 mm Tris-HCl, pH 8.0, 1 mm EDTA buffer. Immune complexes were extracted from the beads with elution buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 1% SDS, and 50 mm NaHCO3) at 65 °C for 4 h.The Re-ChIP assays were performed as described (31Kouskouti A. Talianidis I. EMBO J. 2005; 24: 347-357Crossref PubMed Scopus (213) Google Scholar). Briefly, the eluant of the primary immunocomplex obtained with the first antibody was diluted 10-fold with dilution buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 150 mm NaCl, 1% Triton X-100, and protease inhibitors) and then further subjected to immunoprecipitation with the second antibody. Cross-linking was reversed, and nucleic acids were isolated from the eluates by proteinase K and RNase A treatments followed by phenol extraction and ethanol precipitation. Purified DNA was subjected to PCR using primers covering the AP-1 or SAF-1 DNA-binding regions in the human MMP-1 promoter. PCR products were resolved in a 2% agarose gel and visualized by ethidium bromide staining. The primers used for amplification of the SAF-1 element in human MMP-1 (162 bp) were 5′-GAACTTCAGTCAGTACAGGTGCCGAACAGC-3′ (sense) and 5′-TACGGTCAAAGAGTACTCCATGGTC-3′ (antisense), and those for amplification of the AP-1 element in human MMP-1 (132 bp) were 5′-TCTAATGATTGCCTAGTCTATTCATAGC-3′ (sense) and 5′-CTCCAATATCCCAGCTAGGAAGCTCCCTC-3′ (antisense).Immunoprecipitation and Western Blotting—HTB94 cells, untreated or treated with IL-1β, were lysed using a lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100). The proteins were immunoprecipitated with anti-SAF-1 antibody, anti-c-Jun plus anti-c-Fos antibody, or control nonimmune IgG as indicated. Samples were fractionated in SDS-11% PAGE and electroblotted onto a nitrocellulose membrane. Immunoblotting was performed using anti-c-Fos, anti-c-Jun, or anti-SAF-1 antibody as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody. Bands were detected using a chemiluminescence detection system (Amersham Biosciences).RESULTSSAF-1 Knockdown Affects AP-1-mediated Transcriptional Increase from MMP-1 Gene—To determine the extent of functional cooperation between SAF-1 and AP-1, we used siRNAs directed against SAF-1 and examined the consequence of endogenous SAF-1 inhibition on AP-1-mediated up-regulation of the human MMP-1 promoter. Treatment of HTB-94 cells for 48 h with SAF-1 siRNAs successfully decreased the level of endogenous SAF-1 mRNA as well as protein, as compared with untreated or scrambled siRNA-treated cells (Fig. 1, A and B). Having determined the specificity of SAF-1 siRNAs, we investigated whether knockdown of SAF-1 would show any effect on IL-1β-induced increase of MMP-1 expression. Both RT-PCR analysis (Fig. 1C) and Western blot analysis (Fig. 1D) indicated that reduction of endogenous SAF-1 in response to SAF-1 siRNA treatment of cells substantially reduces the IL-1β-mediated increase of MMP-1 mRNA and protein levels. The specificity of SAF-1 siRNAs on MMP-1 expression was verified by examining MMP-2 protein levels in both untreated and IL-1β-treated cells. There was no detectable change in MMP-2 protein level between scrambled siRNA and SAF-1 siRNA-treated cells during IL-1β treatment or with no treatment (Fig. 1D). In the next experiment, we examined the effect of AP-1 overexpression on the MMP-1-CAT reporter gene in SAF-1-silenced cells. HTB-94 cells were cotransfected with MMP-1(-518/+63)-CAT reporter and an equimolar mixture of c-Fos and c-Jun expression plasmids. Intriguingly, the cells treated with SAF-1 siRNAs failed markedly, by more than 50%, to support c-Fos/c-Jun-mediated induction of MMP-1-CAT reporter expression, whereas cells treated with scrambled siRNAs showed no such inhibitory effect (Fig. 1E). These results suggested a requirement for SAF-1 protein to facilitate AP-1 action and optimal AP-1-mediated transcriptional induction of the MMP-1-CAT reporter.SAF-1 and AP-1 Synergistically Induce MMP-1 Gene Expression—For further assessment of a possible combined regulatory role of SAF-1 and AP-1 in promoting MMP-1 expression, HTB-94 cell were transfected with MMP-1-CAT reporter and expression plasmids of SAF-1 and AP-1. Clearly, simultaneous overexpression of both SAF-1 and AP-1 (c-Fos/c-Jun) expression plasmids synergistically increased MMP-1-CAT reporter transcription (Fig. 2A). The synergy between SAF-1 and AP-1 was not due to any aberrant level of expression from transfected plasmids, as Western blot analysis revealed a dose-dependent increase of SAF-1, c-Fos, and c-Jun proteins in transfected cells (Fig. 2B). These results suggested that SAF-1 and AP-1 act in synergy to induce transcription of the MMP-1 promoter. To better understand how SAF-1 and AP-1 participate in the context of two respective promoter elements of the MMP-1 gene, single and double site mutant promoters were used (Fig. 2C). IL-1β-mediated induction of MMP-1-CAT reporter was partially inhibited when the individual SAF-1 or AP-1 site was mutated. Mutation of both sites resulted in a near complete loss of responsiveness to IL-1β. These results, showing that the participation of IL-1β activated transcription factors at both the SAF-1 and AP-1 sites to promote induction of the MMP-1 gene, were in accord with the previous observation indicating a requirement for both of these proteins.FIGURE 2Synergistic activation of MMP-1 promoter by SAF-1 and AP-1 proteins. A, HTB-94 cells were cotransfected with MMP-1CAT reporter plasmid, increasing concentrations of pcDSAF-1, and an equimolar mixture of pcDc-Fos and pcDc-Jun expression plasmid DNAs as indicated. CAT activity was determined from cells harvested 24 h after transfection. The results shown are representative of three separate experiments. B, detection of SAF-1, c-Jun, and c-Fos expression in transfected cells. Equal amount of cell extracts from HTB-94 cells transfected with increasing concentrations of pcDSAF-1 (lanes 1-3), pcDc-Jun (lanes 4-6), and pcDc-Fos (lanes 7-9) plasmids were immunoblotted with antibodies against SAF-1, c-Jun, and c-Fos as indicated. C, schematic showing physical maps of four reporter plasmids containing either wild-type or specific mutation at either AP-1 or SAF-1 or at both AP-1 and SAF-1 sites as indicated. HTB-94 cells were transfected in duplicate with these reporter plasmids. Following transfection, one set of cells was incubated with IL-1β (500 units/ml) for an additional 24 h. CAT activity was determined. These results represent an average of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Physical Interaction between SAF-1 and AP-1 in Vitro—We contemplated that the cooperativity between SAF-1 and AP-1 might result from a physical interaction between these proteins. To verify this idea, two sets of in vitro experiments, a Far-Western assay and a GST pulldown assay, were performed. The Far-Western assay showed a clear interaction between bacterially expressed His-c-Fos and His-c-Jun proteins with radiolabeled pAR-SAF-1 protein (Fig. 3A, lanes 2 and 3). Radioactive SAF-1 did not interact with the control GST protein (Fig. 3A, lane 1). Input of each protein was examined by Coomassie Blue staining (Fig. 3A, lanes 4-6). To gain more evidence, GST pulldown assays were performed (Fig. 3B). Both bacterially expressed His-c-Fos and His-c-Jun proteins interacted strongly with GST-SAF-1 protein immobilized to a glutathione-Sepharose column and were eluted by a molar excess of glutathione (Fig. 3B, lane 5). However when used alone, His-c-Fos or His-c-Jun protein showed no" @default.
• W2022894345 created "2016-06-24" @default.
• W2022894345 creator A5020111511 @default.
• W2022894345 creator A5022655864 @default.
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• W2022894345 date "2009-01-01" @default.
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