Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 May 15;364(Pt 1):89–99. doi: 10.1042/bj3640089

Identification of an initiator-like element essential for the expression of the tissue inhibitor of metalloproteinases-4 (Timp-4) gene.

David A Young 1, Blaine W Phillips 1, Caroline Lundy 1, Robert K Nuttall 1, Aileen Hogan 1, Gilbert A Schultz 1, Kevin J Leco 1, Ian M Clark 1, Dylan R Edwards 1
PMCID: PMC1222549  PMID: 11988080

Abstract

We have used real-time quantitative reverse transcriptase PCR (TaqMan) to quantify the expression of the four tissue inhibitor of metalloproteinases (Timp) genes in mouse tissues during development and in the adult. Among the four Timp genes, Timp-4 shows the most restricted pattern of expression, with highest RNA levels in brain, heart and testes. These data indicate that in the brain, Timp-4 transcripts are temporally regulated during development, becoming more abundant than those of the other Timps after birth. Cloning of the Timp-4 gene confirmed a five-exon organization resembling that of Timp-2 and Timp-3, and like all Timps, Timp-4 is located within an intron of a synapsin gene. Ribonuclease protection analysis and 5'-rapid amplification of cDNA ends PCR identified multiple transcription starts for Timp-4 from brain and heart mRNA. The promoter region of Timp-4 was functional in transient transfection analysis in mouse C3H10T1/2 fibroblasts, where it directed basal expression that was non-inducible by serum. The TATA-less promoter contains consensus motifs for Sp1 and an inverted CCAAT box upstream of an initiator-like element that is in close proximity to a transcription start site. Mutation of the CCAAT box caused a 2-fold increase in reporter expression. More significantly, mutation of the Sp1 motif or initiator-like element almost completely abolished reporter expression. This first functional characterization of the Timp-4 promoter shows it to be distinct from other members of the Timp family and provides insights into potential mechanisms controlling the tight spatio-temporal expression pattern of the gene.

Full Text

The Full Text of this article is available as a PDF (322.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albright S. R., Tjian R. TAFs revisited: more data reveal new twists and confirm old ideas. Gene. 2000 Jan 25;242(1-2):1–13. doi: 10.1016/s0378-1119(99)00495-3. [DOI] [PubMed] [Google Scholar]
  2. Bigg H. F., Morrison C. J., Butler G. S., Bogoyevitch M. A., Wang Z., Soloway P. D., Overall C. M. Tissue inhibitor of metalloproteinases-4 inhibits but does not support the activation of gelatinase A via efficient inhibition of membrane type 1-matrix metalloproteinase. Cancer Res. 2001 May 1;61(9):3610–3618. [PubMed] [Google Scholar]
  3. Bigg H. F., Shi Y. E., Liu Y. E., Steffensen B., Overall C. M. Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2. J Biol Chem. 1997 Jun 13;272(24):15496–15500. doi: 10.1074/jbc.272.24.15496. [DOI] [PubMed] [Google Scholar]
  4. Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., DeCarlo A., Engler J. A. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197–250. doi: 10.1177/10454411930040020401. [DOI] [PubMed] [Google Scholar]
  5. Blavier L., DeClerck Y. A. Tissue inhibitor of metalloproteinases-2 is expressed in the interstitial matrix in adult mouse organs and during embryonic development. Mol Biol Cell. 1997 Aug;8(8):1513–1527. doi: 10.1091/mbc.8.8.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brew K., Dinakarpandian D., Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000 Mar 7;1477(1-2):267–283. doi: 10.1016/s0167-4838(99)00279-4. [DOI] [PubMed] [Google Scholar]
  7. Butler G. S., Butler M. J., Atkinson S. J., Will H., Tamura T., Schade van Westrum S., Crabbe T., Clements J., d'Ortho M. P., Murphy G. The TIMP2 membrane type 1 metalloproteinase "receptor" regulates the concentration and efficient activation of progelatinase A. A kinetic study. J Biol Chem. 1998 Jan 9;273(2):871–880. doi: 10.1074/jbc.273.2.871. [DOI] [PubMed] [Google Scholar]
  8. Celiker M. Y., Wang M., Atsidaftos E., Liu X., Liu Y. E., Jiang Y., Valderrama E., Goldberg I. D., Shi Y. E. Inhibition of Wilms' tumor growth by intramuscular administration of tissue inhibitor of metalloproteinases-4 plasmid DNA. Oncogene. 2001 Jul 19;20(32):4337–4343. doi: 10.1038/sj.onc.1204508. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dollery C. M., McEwan J. R., Wang M., Sang Q. A., Liu Y. E., Shi Y. E. TIMP-4 is regulated by vascular injury in rats. Circ Res. 1999 Mar 19;84(5):498–504. doi: 10.1161/01.res.84.5.498. [DOI] [PubMed] [Google Scholar]
  12. Doyle G. A., Saarialho-Kere U. K., Parks W. C. Distinct mechanisms regulate TIMP-1 expression at different stages of phorbol ester-mediated differentiation of U937 cells. Biochemistry. 1997 Mar 4;36(9):2492–2500. doi: 10.1021/bi962161e. [DOI] [PubMed] [Google Scholar]
  13. Fager N., Jaworski D. M. Differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mRNA expression during rat central nervous system development. Mech Dev. 2000 Nov;98(1-2):105–109. doi: 10.1016/s0925-4773(00)00437-8. [DOI] [PubMed] [Google Scholar]
  14. Fata J. E., Leco K. J., Moorehead R. A., Martin D. C., Khokha R. Timp-1 is important for epithelial proliferation and branching morphogenesis during mouse mammary development. Dev Biol. 1999 Jul 15;211(2):238–254. doi: 10.1006/dbio.1999.9313. [DOI] [PubMed] [Google Scholar]
  15. Flenniken A. M., Williams B. R. Developmental expression of the endogenous TIMP gene and a TIMP-lacZ fusion gene in transgenic mice. Genes Dev. 1990 Jul;4(7):1094–1106. doi: 10.1101/gad.4.7.1094. [DOI] [PubMed] [Google Scholar]
  16. Gomez D. E., Alonso D. F., Yoshiji H., Thorgeirsson U. P. Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol. 1997 Oct;74(2):111–122. [PubMed] [Google Scholar]
  17. Greene J., Wang M., Liu Y. E., Raymond L. A., Rosen C., Shi Y. E. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem. 1996 Nov 29;271(48):30375–30380. doi: 10.1074/jbc.271.48.30375. [DOI] [PubMed] [Google Scholar]
  18. Groft L. L., Muzik H., Rewcastle N. B., Johnston R. N., Knäuper V., Lafleur M. A., Forsyth P. A., Edwards D. R. Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas. Br J Cancer. 2001 Jul 6;85(1):55–63. doi: 10.1054/bjoc.2001.1854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hernandez-Barrantes S., Shimura Y., Soloway P. D., Sang Q. A., Fridman R. Differential roles of TIMP-4 and TIMP-2 in pro-MMP-2 activation by MT1-MMP. Biochem Biophys Res Commun. 2001 Feb 16;281(1):126–130. doi: 10.1006/bbrc.2001.4323. [DOI] [PubMed] [Google Scholar]
  20. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  21. Inderdeo D. S., Edwards D. R., Han V. K., Khokha R. Temporal and spatial expression of tissue inhibitors of metalloproteinases during the natural ovulatory cycle of the mouse. Biol Reprod. 1996 Sep;55(3):498–508. doi: 10.1095/biolreprod55.3.498. [DOI] [PubMed] [Google Scholar]
  22. Jiang Y., Wang M., Celiker M. Y., Liu Y. E., Sang Q. X., Goldberg I. D., Shi Y. E. Stimulation of mammary tumorigenesis by systemic tissue inhibitor of matrix metalloproteinase 4 gene delivery. Cancer Res. 2001 Mar 15;61(6):2365–2370. [PubMed] [Google Scholar]
  23. Kinoshita T., Sato H., Okada A., Ohuchi E., Imai K., Okada Y., Seiki M. TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads. J Biol Chem. 1998 Jun 26;273(26):16098–16103. doi: 10.1074/jbc.273.26.16098. [DOI] [PubMed] [Google Scholar]
  24. Leco K. J., Apte S. S., Taniguchi G. T., Hawkes S. P., Khokha R., Schultz G. A., Edwards D. R. Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues. FEBS Lett. 1997 Jan 20;401(2-3):213–217. doi: 10.1016/s0014-5793(96)01474-3. [DOI] [PubMed] [Google Scholar]
  25. Leco K. J., Edwards D. R., Schultz G. A. Tissue inhibitor of metalloproteinases-3 is the major metalloproteinase inhibitor in the decidualizing murine uterus. Mol Reprod Dev. 1996 Dec;45(4):458–465. doi: 10.1002/(SICI)1098-2795(199612)45:4<458::AID-MRD8>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  26. Leco K. J., Khokha R., Pavloff N., Hawkes S. P., Edwards D. R. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem. 1994 Mar 25;269(12):9352–9360. [PubMed] [Google Scholar]
  27. Maroulakou I. G., Bowe D. B. Expression and function of Ets transcription factors in mammalian development: a regulatory network. Oncogene. 2000 Dec 18;19(55):6432–6442. doi: 10.1038/sj.onc.1204039. [DOI] [PubMed] [Google Scholar]
  28. Molkentin J. D. The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem. 2000 Dec 15;275(50):38949–38952. doi: 10.1074/jbc.R000029200. [DOI] [PubMed] [Google Scholar]
  29. Nagase H., Woessner J. F., Jr Matrix metalloproteinases. J Biol Chem. 1999 Jul 30;274(31):21491–21494. doi: 10.1074/jbc.274.31.21491. [DOI] [PubMed] [Google Scholar]
  30. Nomura S., Hogan B. L., Wills A. J., Heath J. K., Edwards D. R. Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA. Development. 1989 Mar;105(3):575–583. doi: 10.1242/dev.105.3.575. [DOI] [PubMed] [Google Scholar]
  31. Nothnick W. B., Edwards D. R., Leco K. J., Curry T. E., Jr Expression and activity of ovarian tissue inhibitors of metalloproteinases during pseudopregnancy in the rat. Biol Reprod. 1995 Sep;53(3):684–691. doi: 10.1095/biolreprod53.3.684. [DOI] [PubMed] [Google Scholar]
  32. Nothnick W. B., Soloway P., Curry T. E., Jr Assessment of the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) during the periovulatory period in female mice lacking a functional TIMP-1 gene. Biol Reprod. 1997 May;56(5):1181–1188. doi: 10.1095/biolreprod56.5.1181. [DOI] [PubMed] [Google Scholar]
  33. Olson T. M., Hirohata S., Ye J., Leco K., Seldin M. F., Apte S. S. Cloning of the human tissue inhibitor of metalloproteinase-4 gene (TIMP4) and localization of the TIMP4 and Timp4 genes to human chromosome 3p25 and mouse chromosome 6, respectively. Genomics. 1998 Jul 1;51(1):148–151. doi: 10.1006/geno.1998.5362. [DOI] [PubMed] [Google Scholar]
  34. Overall C. M., King A. E., Sam D. K., Ong A. D., Lau T. T., Wallon U. M., DeClerck Y. A., Atherstone J. Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin carboxyl domain of human gelatinase A by site-directed mutagenesis. The hierarchical role in binding TIMP-2 of the unique cationic clusters of hemopexin modules III and IV. J Biol Chem. 1999 Feb 12;274(7):4421–4429. doi: 10.1074/jbc.274.7.4421. [DOI] [PubMed] [Google Scholar]
  35. Pléau J. M., Esling A., Geutkens S., Dardenne M., Homo-Delarche F. Pancreatic hormone and glutamic acid decarboxylase expression in the mouse thymus: a real-time PCR study. Biochem Biophys Res Commun. 2001 May 18;283(4):843–848. doi: 10.1006/bbrc.2001.4884. [DOI] [PubMed] [Google Scholar]
  36. Pohar N., Godenschwege T. A., Buchner E. Invertebrate tissue inhibitor of metalloproteinase: structure and nested gene organization within the synapsin locus is conserved from Drosophila to human. Genomics. 1999 Apr 15;57(2):293–296. doi: 10.1006/geno.1999.5776. [DOI] [PubMed] [Google Scholar]
  37. Robinson L. L., Sznajder N. A., Riley S. C., Anderson R. A. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human fetal testis and ovary. Mol Hum Reprod. 2001 Jul;7(7):641–648. doi: 10.1093/molehr/7.7.641. [DOI] [PubMed] [Google Scholar]
  38. Smale S. T., Baltimore D. The "initiator" as a transcription control element. Cell. 1989 Apr 7;57(1):103–113. doi: 10.1016/0092-8674(89)90176-1. [DOI] [PubMed] [Google Scholar]
  39. Strongin A. Y., Collier I., Bannikov G., Marmer B. L., Grant G. A., Goldberg G. I. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem. 1995 Mar 10;270(10):5331–5338. doi: 10.1074/jbc.270.10.5331. [DOI] [PubMed] [Google Scholar]
  40. Toth M., Bernardo M. M., Gervasi D. C., Soloway P. D., Wang Z., Bigg H. F., Overall C. M., DeClerck Y. A., Tschesche H., Cher M. L. Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2. J Biol Chem. 2000 Dec 29;275(52):41415–41423. doi: 10.1074/jbc.M006871200. [DOI] [PubMed] [Google Scholar]
  41. Trojanowska M. Ets factors and regulation of the extracellular matrix. Oncogene. 2000 Dec 18;19(55):6464–6471. doi: 10.1038/sj.onc.1204043. [DOI] [PubMed] [Google Scholar]
  42. Tummalapalli C. M., Heath B. J., Tyagi S. C. Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts. J Cell Biochem. 2001;80(4):512–521. doi: 10.1002/1097-4644(20010315)80:4<512::aid-jcb1005>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]
  43. Waterhouse P., Denhardt D. T., Khokha R. Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation. Mol Reprod Dev. 1993 Jul;35(3):219–226. doi: 10.1002/mrd.1080350302. [DOI] [PubMed] [Google Scholar]
  44. Werb Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell. 1997 Nov 14;91(4):439–442. doi: 10.1016/s0092-8674(00)80429-8. [DOI] [PubMed] [Google Scholar]
  45. Wu I., Moses M. A. Molecular cloning and expression analysis of the cDNA encoding rat tissue inhibitor of metalloproteinase-4. Matrix Biol. 1998 Jan;16(6):339–342. doi: 10.1016/s0945-053x(98)90005-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES