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• W2001232517 abstract "Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between −2722 and −7745 allowed for expression in osteoclasts and migrating keratinocytes,i.e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the −2722 to −7745 base pair region. Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between −2722 and −7745 allowed for expression in osteoclasts and migrating keratinocytes,i.e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the −2722 to −7745 base pair region. matrix metalloproteinase 5-bromo-4-chloro-3-indolyl-β-galactopyronoside kilobase pair(s) base pair(s) Mammalian extracellular matrix metalloproteinases (MMPs)1 form a family of related enzymes that are capable of degrading various components of the connective tissue (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2153Crossref PubMed Scopus (3081) Google Scholar, 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1325) Google Scholar, 3Birkedal-Hansen H. Moore W.G.I. 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 (2633) Google Scholar). These proteases are either secreted or membrane-bound and are produced as latent enzymes. They have a conserved Zn2+ binding catalytic site and can be inhibited by specific tissue inhibitors of metalloproteinases (4Charmichael D.F. Sommer A. Thompson R.C. Anderson D.C. Smith C.G. Welgus H.G. Stricklin G.P. Proc. Natl Acad. Sci. U. S. A. 1986; 83: 2407-2411Crossref PubMed Scopus (246) Google Scholar, 5Stetler-Stevenson W.G. Krutzsch H.C. Liotta L.A. J. Biol. Chem. 1989; 264: 17374-17378Abstract Full Text PDF PubMed Google Scholar, 6Goldberg G.I. Marmer B.L. Grant G.A. Eisen A.Z. Wilhelm S. He C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8207-8211Crossref PubMed Scopus (544) Google Scholar, 7Staskus P.W. Masiarz F.R. Pallanck L.J. Hawkes S.P. J. Biol. Chem. 1991; 266: 449-454Abstract Full Text PDF PubMed Google Scholar). In vitro studies imply diverse substrates and functions for these enzymes in vivo. Several genetically distinct enzymes have been identified. Based on in vitro substrate specificities, they are placed in different categories: interstitial collagenases that degrade fibrillar collagens (8Hibbs M.S. Hasty K.A. Seyer J.M. Kang A.H. Mainardi C.L. J. Biol. Chem. 1985; 260: 2493-2500Abstract Full Text PDF PubMed Google Scholar, 9Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar, 10Hasty K.A. Pourmotabbed T.F. Goldberg G. Thompson J.P. Spinella D.G. Stevens R.M. Mainardi C.I. J. Biol. Chem. 1990; 265: 11421-11424Abstract Full Text PDF PubMed Google Scholar); the stromelysins (11Breathnach R. Matrisian L.M. Gesnel M.-C. Staub A. Leroy P. Nucleic Acids Res. 1987; 15: 1139-1151Crossref PubMed Scopus (88) Google Scholar, 12Whitham S.E. Murphy G. Angel P. Rahmsdorf H.-J. Smith B.J. Lyons A. Harris T. Reynolds J.J. Herrlich P. Docherty A.J.P. Biochem. J. 1987; 240: 913-916Crossref Scopus (328) Google Scholar, 13Basset P. Bellocq J.P. Wolf C. Stoll I. Hutin P. Limocher J.M. Podhajcer O.L. Chenard M.P. Rio M.C. Chambon P. Nature. 1990; 348: 699-704Crossref PubMed Scopus (1011) Google Scholar, 14Matrisian L.M. Glaichenhaus N. Gesnel M.-C. Breathnach R. EMBO J. 1985; 4: 1435-1440Crossref PubMed Scopus (227) Google Scholar) with activity against several noncollagenous proteins and collagens with interrupted triple helices; matrilysin that degrades fibronectin, laminin, casein, gelatin, and proteoglycans (2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1325) Google Scholar); macrophage metalloelastase, which degrades elastin (15Shapiro S.D. Griffin G.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Welgus H.G. Senior R.M. Ley T.J. J. Biol. Chem. 1992; 267: 4664-4671Abstract Full Text PDF PubMed Google Scholar, 16Shapiro S.D. Kobayashi D.K. Ley T.J. J. Biol. Chem. 1993; 268: 23824-23829Abstract Full Text PDF PubMed Google Scholar); MMP-2 and MMP-9, which cleave type IV collagen and gelatin (17Salo T. Liotta L.A. Tryggvason K. J. Biol. Chem. 1983; 258: 3058-3063Abstract Full Text PDF PubMed Google Scholar, 18Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar, 19Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 20Huhtala P. Eddy R.L. Fan Y.S. Byers M.G. Shows T.B. Tryggvason K. Genomics. 1990; 6: 554-559Crossref PubMed Scopus (62) Google Scholar, 21Huhtala P. Tuuttila A. Chow L. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar), respectively; and finally a recently described group of the membrane type of matrix metalloproteinases that can activate other metalloproteinases and also degrade matrix proteins (22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 23Will H. Hintzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 24Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Crossref PubMed Scopus (447) Google Scholar). The metalloproteinases are believed to have an important role in normal turnover of extracellular matrix and in remodeling of tissues. Furthermore, they have been shown to be highly expressed in areas of inflammation and tumor invasion. However, the specific roles of the various metalloproteinases in vivo as well as regulation of their genes are still largely unexplored. MMPs 2 and 9, which form a distinct subgroup of MMPs, based on their primary structure and substrate specificity, have been shown to have high activity against gelatin, and they also degrade type IV, V, and VII collagens. However, they do not show high activity against type I collagen, proteoglycan, or laminin. Despite the apparently identical substrate specificity, their temporal and spatial expression in vivo varies extensively, indicating that the two enzymes are required for different purposes. This is also emphasized by the different expression patterns of the two genes (21Huhtala P. Tuuttila A. Chow L. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar). Thus, MMP-2 is primarily expressed in stromal fibroblast-like cells during mouse development (25Reponen P. Sahlberg C. Huhtala P. Hurskainen T. Thesleff I. Tryggvason K. J. Biol. Chem. 1992; 267: 7856-7862Abstract Full Text PDF PubMed Google Scholar), while its expression is insignificant in the stroma of adult mice. This suggests that MMP-2 has a major role in the remodeling of the stromal compartment, in addition to the proposed role in the turnover of basement membrane type IV collagen. In fact, it is possible that the enzyme mainly functions as a stromal gelatinase by removing gelatin derived from degraded fibrillar collagens. In invasive tumors, intense expression of MMP-2 is observed in stromal cells adjacent to the tumor front, not in the tumor cells themselves (26Poulsom R. Pignatelli M. Stetler-Stevenson W.G. Liotta L.A. Wrigth P.A. Jeffery R.E. Longcroft J.M. Rogers L. Stamp G.W.H. Am. J. Pathol. 1992; 141: 389-396PubMed Google Scholar, 27Pyke C. Ralfkiaer E. Huhtala P. Hurskainen T. Danø K. Tryggvason K. Cancer Res. 1992; 52: 1336-1341PubMed Google Scholar, 28Pyke C. Ralfkiaer E. Danö K. Tryggvason K. Am. J. Pathol. 1993; 142: 359-365PubMed Google Scholar, 29Tryggvason K. Höyhtyä M. Pyke C. Breast Cancer Res. Treat. 1993; 24: 209-218Crossref PubMed Scopus (236) Google Scholar). MMP-9 has a completely different expression pattern. By in situ hybridization analysis, its expression has been shown to be almost completely confined to osteoclasts at the site of bone formation during mouse development (30Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Crossref PubMed Scopus (249) Google Scholar). This is the only known example of a highly osteoclast-specific proteinase, which indicates that the enzyme is important for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenases. MMP-9 expression has also been localized to keratinocytes of healing skin wound (31Salo T. Mäkelä M. Autio-Harmainen H. Larjava H. Lab. Invest. 1994; 70: 176-182PubMed Google Scholar), macrophages (27Pyke C. Ralfkiaer E. Huhtala P. Hurskainen T. Danø K. Tryggvason K. Cancer Res. 1992; 52: 1336-1341PubMed Google Scholar,28Pyke C. Ralfkiaer E. Danö K. Tryggvason K. Am. J. Pathol. 1993; 142: 359-365PubMed Google Scholar, 29Tryggvason K. Höyhtyä M. Pyke C. Breast Cancer Res. Treat. 1993; 24: 209-218Crossref PubMed Scopus (236) Google Scholar, 32Mainardi C.L. Hibbs M.S. Hasty K.A. Seyer J.M. Coll. Relat. Res. 1984; 4: 479-492Crossref PubMed Scopus (67) Google Scholar), and trophoblasts of the implanting embryo (33Behrendtsen O. Alexander C.M. Werb Z. Development. 1992; 114: 447-456PubMed Google Scholar, 34Reponen P. Leivo I. Sahlberg C. Apte S.S. Olsen B.R. Thesleff I. Tryggvason K. Dev. Dyn. 1995; 202: 388-396Crossref PubMed Scopus (114) Google Scholar). We have previously shown that no cell-specific expression and regulation of the MMP-9 gene could be achieved through in vitro experiments using transiently transfected cells (35Munaut C. Reponen P. Huhtala P. Kontusaari S. Foidart J.M. Tryggvason K. Ann. N. Y. Acad. Sci. 1994; 732: 369-371Crossref PubMed Scopus (4) Google Scholar). Therefore, to examine the regulation of the MMP-9 gene and, in particular, the regulatory mechanisms of its tissue expression, we have cloned and characterized the mouse gene and studied its regulation using transgenic mice. In this study, we demonstrated that the minimum element(s) required for expression in osteoclasts and migrating keratinocytes reside in the 5′-flanking region of the gene between 2.7 and 7.7 kb upstream of the transcription start site. Mouse genomic libraries cloned in the cosmid pWE15 (Stratagene, catalog no. 95303) and λ Fix phage (Stratagene, catalog no. 46309) were screened using a human MMP-9 cDNA probe (pHG1, Ref.21Huhtala P. Tuuttila A. Chow L. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar) labeled with 32P by random priming. Hybridization was performed in 5× SSC, 5× Denhardt's solution, and 0.1% SDS overnight at 42 °C, after which the filters were washed at a final concentration of 0.1× SSC and 0.1% SDS at 42 °C. The clones were isolated and purified utilizing standard procedures and mapped using restriction endonucleases. The nucleotide sequence was determined by the dideoxynucleotide chain termination procedure (36Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar) using Sequenase or TAQuence DNA sequencing kits (U.S. Biochemical Corp.) and M13 universal primers or specific oligonucleotide primers. Both strands of the gene and its promoter were sequenced following subcloning into pBluescript II SK+/− (Stratagene). Total RNA from 7-day-old mouse skull was isolated by the acid guanidium thiocyanate/phenol chloroform extraction method (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). Primer extension was performed by hybridizing 20 μg of total RNA with an antisense nucleotide primer annealing at positions 117–144 in the cDNA (Ref. 30Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Crossref PubMed Scopus (249) Google Scholar, Fig. 1). The primer was end-labeled by [γ-32P]ATP using T4 polynucleotide kinase (38.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, A. (1989) John Wiley & Sons, Inc., New York.Google Scholar). The reverse transcription reaction was carried out under standard conditions, and the primer-extended products were run on a sequencing gel along with sequencing reactions from the mouse MMP-9 gene using the same oligonucleotide as in the primer extension assay. Promoter-LacZ reporter gene constructs were created using the pKK2480 vector (kindly provided by Mikkel Rohde, University of Copenhagen, Denmark), which contains a multiple cloning site immediately upstream of the LacZ gene. Different length segments of the 5′-flanking region as well as the 5′-end of the MMP-9 gene containing the first exon and intron were excised with appropriate restriction enzymes and used for the construction of MMP-9/LacZ fusion genes. Transgenic animals harboring promoter-reporter gene constructs were generated by injection of the linearized LacZ fusion constructs into pronuclei of fertilized mouse oocytes C57BL/6 × DBA/2 F1 (39Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986: 1-497Google Scholar). Microinjected eggs (15Shapiro S.D. Griffin G.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Welgus H.G. Senior R.M. Ley T.J. J. Biol. Chem. 1992; 267: 4664-4671Abstract Full Text PDF PubMed Google Scholar, 16Shapiro S.D. Kobayashi D.K. Ley T.J. J. Biol. Chem. 1993; 268: 23824-23829Abstract Full Text PDF PubMed Google Scholar, 17Salo T. Liotta L.A. Tryggvason K. J. Biol. Chem. 1983; 258: 3058-3063Abstract Full Text PDF PubMed Google Scholar, 18Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar, 19Collier I.E. Wilhelm S.M. Eisen A.Z. Marmer B.L. Grant G.A. Seltzer J.L. Kronberger A. He C. Bauer E.A. Goldberg G.I. J. Biol. Chem. 1988; 263: 6579-6587Abstract Full Text PDF PubMed Google Scholar, 20Huhtala P. Eddy R.L. Fan Y.S. Byers M.G. Shows T.B. Tryggvason K. Genomics. 1990; 6: 554-559Crossref PubMed Scopus (62) Google Scholar) were then transferred into the oviduct of pseudopregnant NMRI mice, and the mice were allowed to develop to term. At 3 weeks of age, tail DNA was isolated (40Drews R. Drohan W.N. Lubon H. BioTechniques. 1994; 17: 866-867PubMed Google Scholar), and transgenic animals were identified by polymerase chain reaction analysis using two internal primers of the LacZ gene (41Hanley T. Merlie J.P. BioTechniques. 1991; 10: 56PubMed Google Scholar). Mouse embryos from positive mice were recovered at different time points and fixed for 2 h or overnight at 4 °C in 2% paraformaldehyde, 0.2% glutaraldehyde in PBS. They were stained with 5-bromo-4-chloro-3-indolyl-β-galactopyronoside (X-gal) as described by Behringer et al. (42Behringer R.R. Crotty D.A. Tennyson V.M. Brinster R.L. Palmiter R.D. Wolgemuth D.J. Development. 1993; 117: 823-833Crossref PubMed Google Scholar). X-gal-stained mouse embryos and other tissues were rinsed several times in PBS, dehydrated, and embedded in paraffin. Sections of 5–8 μm were stained either by hematoxylin and eosin (43Bancroft J.D. Stevens A. Bancroft J.D. Stevens A. Theory and Practice of Histological Techniques. Third Ed. Churchill Livingstone, Edinburgh, UK1990Google Scholar) or with safranin. Sections were stained for 2–5 min in 0.2% safranin, 1% acetic acid, differentiated for 1–5 s in 95% ethanol, 2–5 s in absolute ethanol, and mounted from xylene. Immunohistochemical staining of paraffin sections (5–10 μm) from skin wounds was carried out by using either the ABComplex horseradish peroxidase kit (DAKO) or the TSA kit (NEN Life Science Products). Deparaffinized sections were treated 5 min. with 0.4% pepsin in 0.01m HCl at 37 °C. Endogenous peroxidase activity was quenched by incubation for 20 min in 3% H2O2, and sections were then incubated with the antiserum raised against cytokeratin (rabbit anti-cytokeratin (Pan), Zymed Laboratories Inc.) for 1.5 h at room temperature. or MMP-9 (rabbit polyclonal antibody kindly provided by P. Carmeliet) overnight at room temperature. For the anti-cytokeratin immunostaining, the sections were washed in PBS, and subsequently, biotinylated Swine anti-rabbit IgG (Boehringer Mannheim) (1:400 dilution) was applied for 30 min at room temperature. After phosphate-buffered saline washes, a 20-min incubation with ABComplex/horseradish peroxidase was carried out (DAKO Code K355). Peroxidase activity was revealed by incubation with the chromogen substrate 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin and eosin. For the MMP-9 immunostaining, peroxidase swine anti-rabbit IgG (DAKO) was applied and followed by tyramide signal amplification (TSA kit; NEN Life Science Products). AEC chromogen substrate (DAKO) was used to detect peroxidase activity. Sections were counterstained with hematoxylin. A mouse MMP-9 cDNA fragment of 324 bp containing SmaI and EcoRI restriction sites from the M92KD-2 cDNA clone (bases 1915–2239) (30Reponen P. Sahlberg C. Munaut C. Thesleff I. Tryggvason K. J. Cell Biol. 1994; 124: 1091-1102Crossref PubMed Scopus (249) Google Scholar) was subcloned into pSP64 ans pSP65 plasmid vectors (Promega). The pSP64 (sense) and pSP65 (antisense) plasmids vectors were linearized withEcoRI and BamHI restriction enzymes, respectively, and the 35S-uridine 5′-triphosphate (1000 mCi/nmol, Amersham Pharmacia Biotech) labeled RNA probes were transcribed using a transcription kit (Promega). The labeled probes were precipitated with ethanol, dissolved in hybridization buffer, and used at 50,000–60,000 cpm/μl. The in situ hybridization was carried out according to Wilkinson and Green (44Wilkinson D.G. Green J. Copp A.J. Cockroft D.L. Postimplantation Mammalian Embryos. Oxford University Press, Oxford1990: 155-171Google Scholar). Prior to hybridization the mouse embryos were stained with X-gal, embedded in paraffin, and sectioned. The specimens were pretreated as described before (45Iivanainen A. Kortesmaa J. Sahlberg C. Morita T. Bergmann U. Thesleff I. Tryggvason K. J. Biol. Chem. 1997; 272: 27862-27868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and hybridized with the probe at 5O °C for 16 h. After washing under high stringency conditions, the sections were dried and dipped in an autoradiographic emulsion (nitro blue tetrazolium-2; Eastman Kodak Co.), exposed for 14 days at 4 °C. After development of the sections, they were stained with hematoxylin and mounted. Screening of the genomic libraries yielded several clones, one of which (cosmid MGC-1 (about 34 kilobases)), contained the entire 7.7-kilobase gene (Fig.1), as well as 3 and 23 kilobases of the 5′- and 3′-end flanking regions, respectively. Another λ phage clone CM-1 contained about 11,000 bp of the 5′-flanking region. Sequencing of exons revealed that the murine gene contains 13 exons, which correspond in size to those of the human gene (Fig. 1, TableI). The only significant differences are exons 9 and 13 which, respectively, contain 54 and 15 base pairs more than in the human gene. Furthermore, introns 1, 2, 3, and 12 in the mouse gene are about half the size of corresponding human introns (21Huhtala P. Tuuttila A. Chow L. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar).Table IExon-intron junctions of the mouse gene for MMP-9Exon/intron numberExon-intron junctionsExon sizeIntron sizebpbp1.......... ATG AGT CCC... ....CTG GCA GAG gtagacagat141 + 19435Met Ser Pro Leu Ala Glu1 472ccatccacag GCA TAC TTG... ....ATC ACA TAC TG gtgagatgtc230223Ala Tyr Leu Ile Thr Tyr Tr(p)48 1243tcacctccag G ATC CAA AAC... ....GGT GTG GCG G gtgagaattc149270aSize determined by polymerase chain reaction with specific oligonucleotides.Tr(p) Ile Gln Asn Gly Val Ala Gl(u)125 1744cctcttgcag AG CAC GGA GAC... ....AAA GGC GTC G gtgagatcct129106Gl(u) His Gly Asp Lys Gly Val Va(l)175 2175ctttctacag TG ATC CCC ACT... ....CCT AGT GAG A gtgagtatgc174176(Va)l Ile Pro Thr Pro Ser Glu Ar(g)218 2756gtcaggtcag GA CTC TAC ACA... ....CCT ACC CGA G gtacctctgc174350(Ar)g Lys Tyr Thr Pro Thr Arg Va(l)276 3337gtctctccag TG GAC GCG ACC... ....CCA GAC CAA G gtgagcgggg177105(Va)l Asp Ala Thr Pro Asp Gln Gl(y)334 3928taccttccag CC TAC AGC CTG... ....TAT CTG TAT G gtgaggctgg1561150aSize determined by polymerase chain reaction with specific oligonucleotides.(Gl)y Tyr Ser Leu Tyr Leu Tyr Gl(y)393 4449ttgtttttag GT CGT GGC TCT... ....TTC AAG GAC GG gtaagcaggg334102(Gl)y Arg Gly Ser Phe Lys Asp Gl(y)445 55510ttcttctcag T TGG TAC TGG... ....TTC TTC TCT G gttagtttgt140270(Gl)y Trp Tyr Trp Phe Phe Ser (Gl)y556 60211tcttccgcag GA CGT CAA ATG... ....CGT GTC TGG AG gtaagagcga151103(Gl)y Arg Gln Met Arg Val Trp Ar(g)603 65212ccttctgcag A TTC GAC TTG... ....CAG TAC CAA G gtgagggctg1041125aSize determined by polymerase chain reaction with specific oligonucleotides.(Ar)g Phe Asp Leu Gln Tyr Gln As(p)653 68713tttcccgtag AC AAA GCC TAT... ....CAG TGC CCT TGA131 + 9433′-UTRbUntranslated region.(As)p Lys Ala Tyr Glu Cys Pro ***688 730Nucleotide sequences at the intron (lowercase letters) and exons (uppercase letters) junctions are shown. The derived amino acid sequence and corresponding position in the polypeptide are displayed below. Amino acids encoded by split codons are listed twice and partially in parentheses. Asterisks indicate translation stop codon.a Size determined by polymerase chain reaction with specific oligonucleotides.b Untranslated region. Open table in a new tab Nucleotide sequences at the intron (lowercase letters) and exons (uppercase letters) junctions are shown. The derived amino acid sequence and corresponding position in the polypeptide are displayed below. Amino acids encoded by split codons are listed twice and partially in parentheses. Asterisks indicate translation stop codon. The initiation site for transcription as determined by primer extension revealed a double start site located 19 and 20 bp upstream of the translated sequence (Fig. 2). Sequencing of about 2800 base pairs of the 5′-flanking region revealed several common promoter elements (Fig. 1). There is a TATA box-like motif TTAAA at positions −30 to −25 but no CCAAT box. There are three GC boxes that may serve as binding sites for the transcription factor Sp1 (46Lee W. Mitchell P. Tjian R. Cell. 1987; 49: 741-752Abstract Full Text PDF PubMed Scopus (1362) Google Scholar) (located at positions −62 to −57, −451 to −446, and −598 to −589). Four AP-1-like binding sites were also identified (−50 to −44, −88 to −80, −472 to −465, and −1080 to −1072). Two of those correspond to similar sequences in the human gene, but sites corresponding to the first one (−50 to −44) and the most upstream one have not been reported in the human gene. Several conserved sequence elements with similarity to the polyoma virus enhancer A-binding protein-3 sites (47Gutman A. Wasylyk B. EMBO J. 1990; 9: 2241-2246Crossref PubMed Scopus (399) Google Scholar) were found in the 5′-flanking sequence (−365 to −360, −479 to −474, −658 to −653, and −901 to −896), as well as in the first intron (Fig. 1). One consensus sequence (5′-CCCCAGGC-3′) for AP-2 (−590 to −483), several microsatellite segments of alternating CA residues, as well as one NF-κB motif (−527 to −519) were also present. A putative tumor growth factor-β1-inhibitory element found in the human gene was absent in the murine promoter. During the course of this work, characterizations of the MMP-9 gene from mouse (48Masure S. Nys G. Fiten P. Van Damme J. Opdenakker G. Eur. J. Biochem. 1993; 218: 129-141Crossref PubMed Scopus (93) Google Scholar) and rabbit (49Fini E. Bartlett J. Matsubara M. Rinehart W. Mody M. Girard M. Rainville M. J. Biol. Chem. 1994; 269: 28620-28628Abstract Full Text PDF PubMed Google Scholar) were published. In order to explore the regulatory mechanisms of the MMP-9 gene, we generated transgenic mice by microinjection of different promoter-reporter gene constructs into fertilized oocytes. A total of six constructs containing different portions of the 5′-end of the gene and the β-galactosidase gene as a reporter were made (Fig. 3). Three constructs, 645-LacZ, 2700-LacZ, and 7700-LacZ, contained 0.65, 2.7, and 7.7 kb of the 5′-flanking region, and three constructs, 645ExIn-LacZ, 2700ExIn-LacZ, and 7700ExIn-LacZ, contained additionally the first exon and intron of the MMP-9 gene in front of the LacZ gene. The constructs containing exon 1 had a mutation in the ATG initiator codon for translation (ATG → ATC) to allow translation of the transcript to start from the ATG methionine initiator codon in the LacZ gene. Intron 1 was included in some of the construct, since it has been shown to contain enhancer elements in other genes such as that for the α1 chain of type I collagen (50Rossi P. Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5590-5594Crossref PubMed Scopus (93) Google Scholar). Three to eight lines of mice were generated with each construct to ensure that the expression pattern obtained with each construct was repeatable. Polymerase chain reaction and Southern analyses were carried out to establish integration of the inserts into the genome, and histochemical analyses with X-gal provided evidence about cell-specific expression patterns of the transgene. Transgenic mice were first generated with constructs containing the minimum promoter, 645-LacZ, and a longer one, 2700-LacZ, containing 2.7 kb of the upstream region. However, mice and mouse embryos made with these constructs did not yield any expression of the LacZ gene in cells that normally express MMP-9 in transgenic embryos or adult tissues. Ectopic expression could be observed in some lines with both constructs, but its pattern was neither uniform nor repeatable in four founder lines analyzed (data not shown). The addition of the 5′-end of the MMP-9 gene, including intron 1, to constructs 645-LacZ and 2700-LacZ (i.e. the 5′-untranslated region and the first exon and intron) did not alter the expression pattern. Consequently, it could be concluded that the first intron does not contain cis-acting elements conferring tissue-specific expression of the endogenous MMP-9 gene. Due to the lack of expression with the constructs described above, we made another one, 7700-LacZ, containing 7.7 kb of the upstream sequence and generated transgenic mice. In contrast, to the shorter constructs, mouse embryos harboring 7700-LacZrevealed expression of the LacZ gene in bones of 14.5–16.5-day-old embryos. For example, at embryonic day 15.5, distinct expression could be observed in the scapula, long bones of fore and hind limbs, ribs, and the lower jaw (Fig.4). Additionally, expression was observed in hair follicles in different mouse lines made with this construct. Construct 7700ExIn-LacZ, containing additionally the first exon and intron, yielded similar expression pattern in bones as 7700-LacZ when analyzed in whole X-gal-stained embryos, with the exception that no expression was present in hair follicles (Fig.4). Only mice made transgenic with constructs containing 7.7 kb of the 5′-flanking region of the MMP-9 gene yielded expression of the LacZ gene in bones as shown in whole embry" @default.
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• W2001232517 date "1999-02-01" @default.
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• W2001232517 title "Murine Matrix Metalloproteinase 9 Gene" @default.
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• W2001232517 doi "https://doi.org/10.1074/jbc.274.9.5588" @default.
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