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• W2022921000 abstract "Murine osteoclast precursors and osteoblasts express the integrin αvβ5, the appearance of which on the cell surface is controlled by the β5, and not the αv, subunit. Here, we show that a 173-base pair proximal region of the β5 promoter mediates β5 basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1–FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a β5 silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate β5 gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity. Murine osteoclast precursors and osteoblasts express the integrin αvβ5, the appearance of which on the cell surface is controlled by the β5, and not the αv, subunit. Here, we show that a 173-base pair proximal region of the β5 promoter mediates β5 basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1–FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a β5 silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate β5 gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity. base pair(s) β-galactosidase kilobase(s) dithiothreitol colony-stimulating factor polymerase chain reaction Recognition of bone matrix proteins by osteoblasts and osteoclast precursors profoundly affects their differentiation and function. Such interactions are mediated principally by integrins, which are heterodimeric transmembrane glycoproteins consisting of α and β chains (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 2.Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar, 3.Yamada K.M. Miyamoto S. Curr. Opin. Cell Biol. 1995; 7: 681-689Crossref PubMed Scopus (589) Google Scholar). The osteoclast is a physiological polykaryon derived by fusion of macrophages in a process apparently requiring attachment of the mononuclear precursors to bone matrix (4.Baron R. Neff L. Tran Van P. Nefussi J.R. Vignery A. Am. J. Pathol. 1986; 122: 363-378PubMed Google Scholar, 5.Fallon M.D. Teitelbaum S.L. Kahn A.J. Lab. Invest. 1983; 49: 159-164PubMed Google Scholar). The integrin αvβ3 is expressed predominantly in mature osteoclasts and plays a critical role in osteoclastic bone resorption (6.Rodan S.B. Rodan G.A. J. Endocrinol. 1997; 154: S47-S56PubMed Google Scholar, 7.Horton M.A. Taylor M.L. Arnett T.R. Helfrich M.H. Exp. Cell Res. 1991; 195: 368-375Crossref PubMed Scopus (216) Google Scholar, 8.Sato M. Sardana M.K. Grasser W.A. Garsky V.M. Murray J.M. Gould R.J. J. Cell Biol. 1990; 111: 1713-1723Crossref PubMed Scopus (160) Google Scholar, 9.Ross F.P. Alvarez J.I. Chappel J. Sander D. Butler W.T. Farach-Carson M.C. Mintz K.A. Robey P.G. Teitelbaum S.L. Cheresh D.A. J. Biol. Chem. 1993; 268: 9901-9907Abstract Full Text PDF PubMed Google Scholar, 10.Teitelbaum S.L. Abu-Amer Y. Ross F.P. J. Cell. Biochem. 1995; 59: 1-10Crossref PubMed Scopus (45) Google Scholar). In contrast, the integrin αvβ5, although structurally related to αvβ3 and sharing many of the same target ligands (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar), is expressed on rodent osteoclast precursors but not on mature bone-resorbing polykaryons (11.Shinar D.M. Schmidt A. Halperin D. Rodan G.A. Weinreb M. J. Bone Miner. Res. 1993; 8: 403-414Crossref PubMed Scopus (60) Google Scholar). Murine osteoclast precursors utilize integrin αvβ5 and not αvβ3 for attachment to matrix (12.Inoue M. Namba N. Chappel J. Teitelbaum S.L. Ross F.P. Mol. Endocrinol. 1998; 12: 1955-1962Crossref PubMed Google Scholar). Given these facts, it is not surprising that αvβ3and αvβ5 levels rise and fall, respectively, during osteoclast differentiation (12.Inoue M. Namba N. Chappel J. Teitelbaum S.L. Ross F.P. Mol. Endocrinol. 1998; 12: 1955-1962Crossref PubMed Google Scholar). Osteoblasts express a variety of integrins (13.Clover J. Dodds R.A. Gowen M. J. Cell Sci. 1992; 92: 267-271Crossref Google Scholar, 14.Grzesik W.J. Robey P.G. J. Bone Miner. Res. 1994; 9: 487-496Crossref PubMed Scopus (326) Google Scholar, 15.Hughes D.E. Salter D.M. Dedhar S. Simpson R. J. Bone Miner. Res. 1993; 93: 527-533Google Scholar, 16.Sinha R.K. Tuan R.S. Bone. 1996; 18: 451-457Crossref PubMed Scopus (178) Google Scholar, 17.Gronthos S. Stewart K. Graves S.E. Hay S. Simmons P.J. J. Bone Miner. Res. 1997; 12: 1189-1197Crossref PubMed Scopus (215) Google Scholar) that interact with bone matrix proteins to prompt commitment to the bone synthesizing phenotype. Thus, human osteoblasts adhere to vitronectin in an Arg-Gly-Asp (RGD)-dependent manner (14.Grzesik W.J. Robey P.G. J. Bone Miner. Res. 1994; 9: 487-496Crossref PubMed Scopus (326) Google Scholar) and an RGD peptide inhibits matrix mineralization in vitro (18.Moursi A.M. Damsky C.H. Lull J. Zimmerman D. Doty S.B. Aota S. Globus R.K. J. Cell Sci. 1996; 109: 1369-1380Crossref PubMed Google Scholar, 19.Gronowicz G.A. Derome M.E. J. Bone Miner. Res. 1994; 9: 193-201Crossref PubMed Scopus (69) Google Scholar). Whereas osteoblasts at the bone surface express high levels of α5β1, αvβ3 and αvβ5 (14.Grzesik W.J. Robey P.G. J. Bone Miner. Res. 1994; 9: 487-496Crossref PubMed Scopus (326) Google Scholar, 20.Hultenby K. Reinholt F.P. Heinegard D. Eur. J. Cell Biol. 1993; 62: 86-93PubMed Google Scholar), adhesion of osteoblasts to vitronectin is mediated specifically by αvβ5. 1S.-L. Cheng, unpublished data. Sp1, a ubiquitously expressed transcription factor containing three Zinc finger motifs, Cys2-His2, which bind the consensus sequence GGGGCGGGGC (21.Letovsky J. Dynan W.S. Nucleic Acids Res. 1989; 17: 2639-2653Crossref PubMed Scopus (182) Google Scholar), regulates a various genes in a constitutive or an inducible manner (22.Lania L. Majello B. De Luca P. Int. J. Biochem. Cell Biol. 1997; 29: 1313-1323Crossref PubMed Scopus (263) Google Scholar, 23.Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (391) Google Scholar). Three Sp1-related proteins, Sp2, Sp3, and Sp4, have also been cloned (22.Lania L. Majello B. De Luca P. Int. J. Biochem. Cell Biol. 1997; 29: 1313-1323Crossref PubMed Scopus (263) Google Scholar). Although all four molecules have similar structural features, including highly conserved DNA binding domains, Sp1, Sp3, and Sp4 share closest homology (22.Lania L. Majello B. De Luca P. Int. J. Biochem. Cell Biol. 1997; 29: 1313-1323Crossref PubMed Scopus (263) Google Scholar). Although Sp3 is widely expressed and can activate or repress gene expression (24.Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (204) Google Scholar, 25.Majello B. De Luca P. Lania L. J. Biol. Chem. 1997; 272: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), Sp4 is solely a transcriptional activator, found only in specific brain cells (26.Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (525) Google Scholar). PU.1, a member of the E twenty-six family of transcription factors, is expressed in macrophages, B cells, mast cells, and neutrophils (27.Zhang D.E. Hohaus S. Voso M.T. Chen H.M. Smith L.T. Hetherington C.J. Tenen D.G. Curr. Top. Microbiol. Immunol. 1996; 211: 137-147PubMed Google Scholar,28.Moreau-Gachelin F. Biochim. Biophys. Acta. 1994; 1198: 149-163PubMed Google Scholar). E twenty-six family proteins are characterized by a DNA binding domain that recognizes purine-rich sequences, typically containing a 5′-GGAA-3′ core. PU.1 is necessary for both normal myelopoiesis (29.Henkel G.W. McKercher S.R. Yamamoto H. Anderson K.L. Oshima R.G. Maki R.A. Blood. 1996; 88: 2917-2926Crossref PubMed Google Scholar,30.McKercher S.R. Torbett B.E. Anderson K.L. Henkel G. Vestal D.J. Klemsz M. Feeney A.J. Wu G.E. Paige C.J. Maki R.A. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (934) Google Scholar) and osteoclast differentiation (31.Tondravi M.M. McKercher S.R. Anderson K. Erdmann J.M. Quiroz M. Maki R. Teitelbaum S.L. Nature. 1997; 386: 81-84Crossref PubMed Scopus (454) Google Scholar). Given that the β5 integrin subunit is expressed in both osteoclast precursors and osteoblasts, we turned to the molecular mechanism by which the gene is regulated in these two important cell types. Taking advantage of our recently cloned murine integrin β5 gene promoter (32.Feng X. Teitelbaum S.L. Quiroz M.E. Towler D.A. Ross F.P. J. Biol. Chem. 1999; 274: 1366-1374Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), we identified three Sp1/Sp3 sites in a 173-bp2 β5proximal promoter region and established that they mediate basal transcription in osteoblasts. Each of the Sp1/Sp3 sites plays a different role in this process. The first Sp1/Sp3 site (–53 to –48) acts as weak enhancer, the second site (–26 to –17) serves as a strong enhancer, and the third (+29 to +36) represses transcription. The same three Sp1/Sp3 regulate the β5 promoter in macrophages in a manner similar to that in osteoblasts. In contrast to its typical enhancer function, a downstream PU.1 site (+73 to +84), which is functional only in osteoclast precursors, serves as a silencer of the β5 promoter. pGL3-basic plasmid, a promoterless luciferase construct, was purchased from Promega (Madison, WI). A 1-kb β5 promoter-luciferase construct (pGL3–1kb(+)) and its deletion mutants (pGL3(–796), pGL3(–633), pGL3(–483), pGL3(–340), pGL3(–172), and pGL3(–63)) were prepared as in our previous study (32.Feng X. Teitelbaum S.L. Quiroz M.E. Towler D.A. Ross F.P. J. Biol. Chem. 1999; 274: 1366-1374Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, in pGL3–1kb(+), a nearly 1-kb proximal region (from –875 to +110) of the β5 promoter containing the transcriptional start site (which was designated +1) was placed, in the sense orientation to the luciferase gene, in the pGL3-basic plasmid. All mutants were made by deleting the 5′ end of the 1-kb promoter fragment in the pGL3–1kb(+) in a progressive fashion (32.Feng X. Teitelbaum S.L. Quiroz M.E. Towler D.A. Ross F.P. J. Biol. Chem. 1999; 274: 1366-1374Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Therefore, these constructs contain promoter fragments with the same 3′ ends (+110) and different 5′ ends (the locations of these different 5′ ends are indicated by the numbers in the parenthesis of these mutant constructs). In the current study, we made the point mutation constructs (Mu-1, Mu-2, Mu-3, Mu-12, Mu-13, Mu-23, Mu-123, and Mu-4) as detailed below, under “Site-directed Mutagenesis.” The mouse macrophage cell line J774 (J774A.1) and a mouse pre-osteoblast cell line, MC3T3-E1, were cultured in minimum essential medium α modification (Sigma) containing 10% heat-inactivated fetal bovine serum from Life Technologies, Inc. Cells were transfected with LipofectAMINE PlusTM reagent (Life Technologies, Inc.) as follows. One day prior to transfection, J774 cells were scraped off tissue culture dishes with Cell Lifters (Costar, Corning, NY), whereas MC3T3-E1 cells were lifted by trypsinization. The cells were counted and replated in six-well plates (5 × 105 cells/well). The next day, every well was treated with a mixture prepared as follows. 1 μg of reporter plasmid and 0.1 μg of cytomegalovirus-β-galactosidase (β-gal) plasmid were diluted with Opti-MEM (Life Technologies, Inc.) and mixed with the Plus reagent to form a pre-complex at room temperature for 15 min. The pre-complex was then mixed and incubated with the LipofectAMINE reagent (previously diluted with Opti-MEM) at room temperature for 15 min to form DNA Plus LipofectAMINE, which was added to each well. The complex was mixed gently into the medium and incubated at 37 °C at 5% CO2 for 4 h. Then, the medium was replaced with minimum essential medium α modification containing 10% fetal bovine serum and incubated at 37 °C at 5% CO2. 24 h later, cells were washed with phosphate-buffered saline twice, lysates were prepared, and luciferase activity was measured using the luciferase assay kits from Promega (Madison, WI), with normalization to β-gal activity measured separately. Nuclear extracts used for both DNase I footprinting assays and gel shift assays were prepared as follows. Both cell types cultured until they reached confluence were washed three times with cold phosphate-buffered saline and incubated with 20 ml of phosphate-buffered saline containing 5 mmEDTA and 5 mm EGTA for 30 min on ice. Cells from two plates were scraped off the dishes with rubber policemen, pooled, spun down, resuspended in 1.5 ml of cold phosphate-buffered saline, and transferred to 2-ml microcentrifuge tubes. The cells were pelleted in a microcentrifuge for 30 s, media were removed, and the cells were resuspended in 500 μl of hypotonic lysis buffer (10 mmHepes-KOH, pH 7.9, 10 mm KCl, 1.5 mmMgCl2, 0.5 mm DTT, 0.5 mmphenylmethylsulfonyl fluoride; DTT and phenylmethylsulfonyl fluoride were added freshly). Cells were lysed for 15 min on ice, at which time 32 μl of 10% Nonidet P-40 was added to the suspension, followed by vortexing the tube for 15 s and incubating on ice for 10 min. Nuclei were spun down and resuspended in 100 μl of nuclear extraction buffer (20 mm Hepes-KOH, pH 7.9, 420 mm NaCl, 1.2 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm DTT, 0.5 mmphenylmethylsulfonyl fluoride, 0.5 mm 4-(2-aminoethyl) benzenesulfonyl fluoride, 5 μg/ml pepstatin, and 5 μg/ml leupeptin). DTT, phenylmethylsulfonyl fluoride, 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin, and leupeptin were added freshly to the buffer. The extract was kept for 20 min on ice and spun down in a microcentrifuge. The supernatant (nuclear extract) was aliquoted, quickly frozen in dry ice/ethanol bath, and stored at −70 °C. Protein concentration of nuclear extracts was determined using the Micro BCA kit (Pierce). Two DNA probes,BssHI*-BglII andBssHI-HindIII* (the asterisk indicates the site of 32P labeling), were prepared for DNase I footprinting assays as follows. Reporter plasmid pGL3–1kb(+), which contains a 1-kb β5 promoter fragment (–875 to +110), was cut with BssHI and HindIII to generate a proximal promoter fragment BssHI-HindIII (see Fig.2 A for details), which was end-labeled with 32P using Klenow fragment (Life Technologies, Inc.). To prepare single-end-labeled probes, the double-end-labeled fragment (BssHI*-HindIII*) was extracted with phenol, precipitated with ethanol, resuspended in ddH2O, and digested with BglII at 37 °C for 2 h. The large and small fragments (BssHI*-BglII andBglII-HindII*I) were separated by applying the digestion mixture to Quick SpinTM Sephadex Column G-50 (Roche Molecular Biochemicals). The purified large fragmentBssHI*-BglII has only one end labeled (BssHI). To prepare probeBssHI-HindIII*, pGL3–1kb(+) was cut withPstI and HindIII to obtain the fragmentPstI-HindIII (see Fig. 2 A). The fragment (PstI-HindIII) was labeled with32P, redigested with BssHI, and purified using Quick SpinTM Sephadex Column G-50 as described above. The resulting fragment BssHI-HindIII*, was labeled only at its HindIII end. DNase I footprinting experiments were performed as described by Kucharczuk and Goldhamer (33.Kucharczuk K.L. Goldhamer D.J. Methods Cell Biol. 1997; 52: 439-472Crossref PubMed Google Scholar). In short, 1 μl (around 1 × 105cpm) of the one-end-labeled DNA fragment (BssHI-BglII) or (BssHI-HindIII) was incubated with 10 μg of nuclear extract, prepared as described above, in 25 μl of binding reaction solution (containing 12 mm Tris-Cl, pH 8.0, 1 mm MgCl, 5 mm NaCl, 1 mmCaCl2, 0.1 μg/ul bovine serum albumin, 0.1 mmDTT, 5% glycerol, 50 mm KCl; 40 ng/μl poly(dI·dC), and 2% polyvinyl alcohol) on ice for 60 min. For the bovine serum albumin control, 2.5 μg of bovine serum albumin was used instead of 10 μg of nuclear extract in binding reaction containing 60 mminstead of 50 mm KCl. Three binding reactions for the experimental sample and three control reactions were set up to allow for three different concentrations. DNase I stock (10 unit/μl) was purchased from Roche Molecular Biochemicals. After a 60-min incubation on ice, the experimental binding reactions were treated with 1 μl of 1/10, 1/20, or 1/40 dilution of the stock for 2 min, and the bovine serum albumin controls were treated with 1 μl of 1/400, 1/800, and 1/1600 dilution of the stock for 2 min. DNase I-treated binding reactions were digested with proteinase K, followed by extraction with phenol. The DNA fragments were precipitated with ethanol and then separated by 6% sequencing gel. An unrelated sequencing reaction was used as a size marker. Oligonucleotides (oligos) used for gel shift assays were synthesized by Life Technologies, Inc. and end-labeled with 32P by T4 polynucleotide kinase (Life Technologies, Inc.). 1 × 105 cpm probe was incubated with 2 μg of nuclear extracts (prepared as described above) in a 20-μl volume of binding reaction (10 mm Tris-Cl, pH 7.5, 100 mm NaCl, 10% glycerol, 50 ng/ml poly(dI·dC)) on ice for 20 min. In competition experiments, a 20× or 100× excess amount of unlabeled competitors was premixed with 1 × 105cpm of labeled probe before being added to the binding mixture. The binding reaction was then allowed to proceed for 20 min on ice. In supershift experiments, a 1 × 105 cpm probe was incubated with 2 μg of nuclear extracts in a 20-μl volume of binding reaction for 20 min on ice, at which time 2 μl of nonimmune serum or 2 μl of specific antibodies (2 μg/ul) was added, followed by incubation on ice for an additional 30 min. All binding mixtures were separated, using 0.5× TBE buffer as the running buffer, at 4 °C at 100 V for 3.5 h by 4–20% gradient TBE gels (Novex, San Diego, CA) in a Novex XCell IITM minicell electrophoresis system. The gels were transferred to 3M blotting paper, dried, and exposed to film with an intensifying screen at −70 °C. Antibodies (anti-Sp1, anti-Sp3, and anti-PU.1) and the nonimmune serum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Point mutations were introduced in the context of pGL3(–63), which contains 173-bp proximal β5 promoter fragment (from –63 to +110), using a QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligos used to mutate the Sp1/Sp3 site within FP1 were 5-GGTCCCGGTGCAGttCGGAGCTGG-3′ and 5′-CCAGCTCCGaaCTGCACCGGGACC-3′. Oligos used to mutate the Sp1/Sp3 site within FP2 were 5′-GAGCTCGCCCCGaaCCGTCCCGCC-3′ and 5′-GGCGGGACGGttCGGGGCGAGCTC-3′. Oligos used to mutate the Sp1/Sp3 site within FP3 were 5′-GCAGGAGAGGGttGAGGAGAAAGC-3′ and 5′-GCTTTCTCCTCaaCCCTCTCCTGC-3′. Oligos used to mutate the PU.1 site within FP4 were 5′-CTGGCGGCCGAGGAAAAAcccAAGGGTCTCCGAGAGTAG-3′ and 5′-CTACTCTCGGAGACCCTTgggTTTTTCCTCGGCCGCCAG-3′, with the lowercase letters indicating the mutation sites. These oligonucleotides were purchased from Life Technologies, Inc. and purified by polyacrylamide gel electrophoresis. PCRs were performed in a 50-μl volume withPfu polymerase (Stratagene, La Jolla, CA), 10 ng of DNA template, and 125 ng of each oligo using the following conditions: 95 °C for 30 s, 1 cycle; 95 °C for 30 s, 55 °C for 1 min and 68 °C for 12 min, 16 cycles; and 4 °C. The PCR was treated with DpnI (10 units) for 60 min at 37 °C. XL1-Blue supercompetent cells were transformed with theDpnI-treated PCR mixture as described in the instruction manual and plated on ampicillin plates. Plasmids were prepared from individual colonies and sequenced to confirm the correctness of the introduced mutations. All single-site mutants (Mu-1, Mu-2, Mu-3, and Mu-4) were generated using pGL3(–63) as template, the respective pair of oligos, and the PCR condition described above. The double-site mutants (Mu-12, Mu-13, and Mu-23) were made by performing a second round of PCR using the single-site mutants as template and the appropriate pairs of oligos. The generation of the triple-site mutant (Mu-123) was just an extension of the same approach, using a third set of oligos to perform another round of PCR. Sequence analysis was performed using the Genetic Computer Group (Madison, WI) sequence analysis software. Osteoclast precursors and osteoblasts express the integrin αvβ5 (11.Shinar D.M. Schmidt A. Halperin D. Rodan G.A. Weinreb M. J. Bone Miner. Res. 1993; 8: 403-414Crossref PubMed Scopus (60) Google Scholar, 12.Inoue M. Namba N. Chappel J. Teitelbaum S.L. Ross F.P. Mol. Endocrinol. 1998; 12: 1955-1962Crossref PubMed Google Scholar, 14.Grzesik W.J. Robey P.G. J. Bone Miner. Res. 1994; 9: 487-496Crossref PubMed Scopus (326) Google Scholar, 20.Hultenby K. Reinholt F.P. Heinegard D. Eur. J. Cell Biol. 1993; 62: 86-93PubMed Google Scholar). We previously cloned the integrin β5 promoter and observed that a 173-bp proximal β5 promoter fragment, pGL3(–63) (–63 to +110), is capable of mediating β5 gene transcription in the myeloid cell line FDCP-Mac11(32), suggesting that this region contains basal transcription factor binding sites. To pursue this observation, we turned to the macrophage cell line, J774, which is more easily transfected and in which pGL3(–63) also conferred basal promoter activity (Fig. 1 A). Similar results were obtained with the murine osteoblast cell line MC3T3-E1 (Fig. 1 B). To identify potential transcription factor binding sites by which the 173-bp proximal promoter region controls β5transcription, we performed in vitro DNase I footprinting assays with nuclear extracts of J774 or MC3T3-E1 cells. Four restriction sites (PstI, BssHI, BglII, and HindIII) flanking the 173-bp region were used to prepare two probes (Fig. 2 A). ProbeBssHI-HindIII* was labeled at HindIII end, and probe BssHI*-BglII was labeled atBssHI end. Using probe BssHI-HindIII* (Fig. 2 B), four regions (FP1–FP4) were protected by J774 nuclear proteins, whereas MC3T3 extracts protected only three sites (FP1–FP3). To confirm this result, we repeated the footprinting assays with the probe BssHI*-BglII (Fig. 2 C). Similar results were obtained with nuclear extracts prepared from mouse primary bone marrow macrophages or osteoblasts (data not shown). To determine whether these protected genomic sequences bind nuclear proteins, we synthesized four oligonucleotides (FP1–FP4), each containing one of the protected sequences (Fig.3 A). In gel shift assays (Fig.3 B), oligos FP1, FP2, and FP3 gave rise to slowly (A) and quickly (B) migrating major bands with both J774 and MC3T3-E1 nuclear extracts. As demonstrated by subsequent studies (Figs. Figure 4, Figure 5, Figure 6), A comprises two or three bands, depending on which oligo was used for binding. Oligo FP2 also yielded a number of minor, high mobility bands bands (band C) from J774 nuclear extracts and one (band D) from MC3T3 nuclear extracts. Oligo FP3 generated a minor band (band E) with high mobility only from MC3T3 nuclear extracts. 3Although all minor bands were consistently detectable on long exposure, they are not always visible in some later figures. Consistent with the footprinting experiments (Fig. 2), oligo FP4 binds a nuclear protein (band F) only from J774 cells. Thus, several cis-elements within the proximal promoter region may mediate basal transcription of the β5 gene.Figure 6Identification of point mutations in Sp1 sites in FP1, FP2, and FP3 and in PU.1 site in FP4 that mediate binding. A, sequences of wild type (uppercase) and mutant (m) FP1 (lowercase), FP2, and FP3. The Sp1-like consensus sequences in each oligo isboxed. B, gel shift competition assays with the oligos described in A. Gel shift assays were performed with J774 nuclear extracts using oligo FP1 (lanes 1–5), FP2 (lanes 6–10), and FP3 (lanes 11–15) as probes, respectively. For each probe, the bands were competed with excess of unlabeled wild type oligos (lanes 2 and 3 for FP1, lanes 7 and 8 for FP2, and lanes 12 and 13 for FP3) but not mutant (m) oligos (lanes 4 and 5 for FP1, lanes 9 and10 for FP2, and lanes 14 and 15 for FP3). C, sequences of wild type and mutant FP4 oligonucleotides. In all oligos, the sequence identified by the footprinting assays that contains a putative PU.1 site isunderlined. A PU.1 consensus sequence located partially in the protected region is indicated by asterisks below the sequence. Mutated nucleotides are shown in lowercase. D, gel shift competition assays with the six mutant FP4 oligos. Gel shift assays were performed using labeled wild type FP4 oligo and J774 nuclear extracts (lane 1) with or without mutants M1–M6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4FP1, FP2, and FP3 bind Sp1 family members, and FP4 recognizes PU.1 from J774 nuclear extracts. A,gel shift assays using labeled oligo FP1 as probe were competed with unlabeled oligo FP1 (lanes 2 and 3), an Sp1 consensus sequence (5′-ATTCGATCGGGGCGGGGCGAGC-3′) (lanes 4 and 5), and a mutated Sp1 consensus sequence (5′-ATTCGATCGGttCGGGGCGAGC-3′) (lanes 6and 7). B, the experiment shown in Awas repeated with oligo FP2. C, the experiment shown inA was repeated with oligo FP3. D, competition assays with FP4 as probe. Gel shift assays using labeled oligo FP4 as probe were competed with excess of cold oligo FP4 (lanes 2and 3), a PU.1 consensus sequence (5′-GGGCTGCTTGAGGAAGTATAAGAAT-3′) (lanes 4 and5), and a mutated PU.1 consensus sequence (5′-GGGCTGCTTGAGagaGTATAAGAAT-3′) (lanes 6 and7).View Large Image Figure ViewerDownload Hi-res image Download (PPT) FP1, FP2, and FP3 each contain potential Sp1 sites. A canonical PU.1 consensus sequence is located partially within FP4, which also includes a noncanonical PU.1 binding region (34.Pahl H.L. Scheibe R.J. Zhang D.E. Chen H.M. Galson D.L. Maki R.A. Tenen D.G. J. Biol. Chem. 1993; 268: 5014-5020Abstract Full Text PDF PubMed Google Scholar) (Fig.3 A). To determine whether the proteins from J774 nuclear extracts binding to FP1–FP3 include Sp1 family members and those binding to FP4 include PU.1, respectively. we performed gel shift/competition assays. As seen in Fig. 4 A, 32P-labeled FP1 oligos give rise to four bands (a–d, lane 1), each being eliminated by excess unlabeled probe (lanes 2 and 3). A 22-bp oligo containing an Sp1 consensus sequence abolished bands a, b, and c (lanes 4 and 5). When mutated, the consensus Sp1 oligo had no effect on bands a, b, or c (lanes 6 and 7), suggesting that they are Sp1-related proteins. Given that band d was not affected by excess unlabeled wild type Sp1 oligo, it probably represents a nuclear protein that is not a member of the Sp1 family. Gel shift analysis using 32P-labeled FP2 oligo yielded three major bands, a, b, and c, as well as several minor bands, all eliminated by excess unlabeled FP2, (Fig. 4 B, lanes 1 and2). Wild type, but not mutated, Sp1 oligo abrogated all bands. As seen in Fig. 4 C, FP3 also specifically bound Sp1 related proteins. FP4 probe, incubated with J774 nuclear extract, yielded one band (Fig. 4 D, lane 1, e), diminished by unlabeled FP4 (lanes 2 and 3). Wild type (lanes 4 and 5) but not mutated (lanes 6 and 7) PU.1 oligo also abolished the band. These experiments suggest, but do not prove, that FP1, FP2, and FP3 recognize Sp1-related transcription factors (Fig. 4, A–C, bands a, b, and c) and that FP4 contains a PU.1 binding site. FP1 also bound a slowly migrating protein not related to the Sp1 family, whereas FP2 yields rapidly migrating proteins. These moieties are either novel members of the Sp1 family or bind to the oligo in a manner requiring concomitant association of Sp1. To confirm the identity of putative Sp1, Sp3, and PU.1 proteins, we performed supershift assays, using appropriate antibodies. Whereas nonimmune serum had no effect on bands generated with probe FP1 (Fig.5 A, lane 2), anti-Sp1 antibody supershifted the lowest mobility species (lane 3), corresponding to band a in Fig. 4 A. Anti-Sp3 antibody, in turn, supershifted two bands (lane 4), corresponding to bands b and c in Fig.4 A. The fourth band, d, was not supershifted by either antibody, indicating that it represents a non-Sp1/Sp3 protein. Similar results were obtained using oligo FP2 (Fig. 5 B, lanes 1–4)" @default.
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• W2022921000 title "Sp1/Sp3 and PU.1 Differentially Regulate β5Integrin Gene Expression in Macrophages and Osteoblasts" @default.
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