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. 1998 Mar 15;101(6):1478–1487. doi: 10.1172/JCI1584

Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis.

A H Baker 1, A B Zaltsman 1, S J George 1, A C Newby 1
PMCID: PMC508704  PMID: 9502791

Abstract

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related secreted proteins that limit matrix metalloproteinase (MMP) activity and also have direct effects on cell growth. We used the highly efficient adenoviral delivery system to overexpress individual TIMPs from the cytomegalovirus immediate early promoter in rat aortic smooth muscle cells. Overexpression of TIMP-1, -2, or -3, or a synthetic MMP inhibitor similarly inhibited SMC chemotaxis and invasion through reconstituted basement membrane. TIMP-1 overexpression did not effect cell proliferation. By contrast, TIMP-2 caused a dose-dependent reduction in proliferation, an effect not mimicked by a synthetic MMP inhibitor. TIMP-3 overexpression induced DNA synthesis, and promoted SMC death by apoptosis, a phenotype reproduced by adding TIMP-3 to uninfected cells, but not by a synthetic MMP inhibitor. Our study is the first to compare systematically the effect of overexpression of three TIMPs in any cell. We found similar effects on invasion mediated by inhibition of MMP activity, but widely divergent effects on proliferation and death through actions of TIMP-2 and -3 independent of MMP inhibition. These findings have important implications for the physiological roles of TIMPs and their use in gene therapy.

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Selected References

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  1. Baker A. H., Wilkinson G. W., Hembry R. M., Murphy G., Newby A. C. Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells. Matrix Biol. 1996 Dec;15(6):383–395. doi: 10.1016/s0945-053x(96)90158-4. [DOI] [PubMed] [Google Scholar]
  2. Bendeck M. P., Irvin C., Reidy M. A. Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury. Circ Res. 1996 Jan;78(1):38–43. doi: 10.1161/01.res.78.1.38. [DOI] [PubMed] [Google Scholar]
  3. Bendeck M. P., Zempo N., Clowes A. W., Galardy R. E., Reidy M. A. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994 Sep;75(3):539–545. doi: 10.1161/01.res.75.3.539. [DOI] [PubMed] [Google Scholar]
  4. Bennett M. R., Anglin S., McEwan J. R., Jagoe R., Newby A. C., Evan G. I. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest. 1994 Feb;93(2):820–828. doi: 10.1172/JCI117036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bian J., Wang Y., Smith M. R., Kim H., Jacobs C., Jackman J., Kung H. F., Colburn N. H., Sun Y. Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3. Carcinogenesis. 1996 Sep;17(9):1805–1811. doi: 10.1093/carcin/17.9.1805. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Docherty A. J., Lyons A., Smith B. J., Wright E. M., Stephens P. E., Harris T. J., Murphy G., Reynolds J. J. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature. 1985 Nov 7;318(6041):66–69. doi: 10.1038/318066a0. [DOI] [PubMed] [Google Scholar]
  8. Fabunmi R. P., Baker A. H., Murray E. J., Booth R. F., Newby A. C. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996 Apr 1;315(Pt 1):335–342. doi: 10.1042/bj3150335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fariss R. N., Apte S. S., Olsen B. R., Iwata K., Milam A. H. Tissue inhibitor of metalloproteinases-3 is a component of Bruch's membrane of the eye. Am J Pathol. 1997 Jan;150(1):323–328. [PMC free article] [PubMed] [Google Scholar]
  10. Forough R., Koyama N., Hasenstab D., Lea H., Clowes M., Nikkari S. T., Clowes A. W. Overexpression of tissue inhibitor of matrix metalloproteinase-1 inhibits vascular smooth muscle cell functions in vitro and in vivo. Circ Res. 1996 Oct;79(4):812–820. doi: 10.1161/01.res.79.4.812. [DOI] [PubMed] [Google Scholar]
  11. Fridman R., Fuerst T. R., Bird R. E., Hoyhtya M., Oelkuct M., Kraus S., Komarek D., Liotta L. A., Berman M. L., Stetler-Stevenson W. G. Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions. J Biol Chem. 1992 Aug 5;267(22):15398–15405. [PubMed] [Google Scholar]
  12. Galis Z. S., Sukhova G. K., Lark M. W., Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994 Dec;94(6):2493–2503. doi: 10.1172/JCI117619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. George S. J., Zaltsman A. B., Newby A. C. Surgical preparative injury and neointima formation increase MMP-9 expression and MMP-2 activation in human saphenous vein. Cardiovasc Res. 1997 Feb;33(2):447–459. doi: 10.1016/s0008-6363(96)00211-8. [DOI] [PubMed] [Google Scholar]
  14. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  15. Hayakawa T., Yamashita K., Ohuchi E., Shinagawa A. Cell growth-promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2). J Cell Sci. 1994 Sep;107(Pt 9):2373–2379. doi: 10.1242/jcs.107.9.2373. [DOI] [PubMed] [Google Scholar]
  16. Howard E. W., Bullen E. C., Banda M. J. Preferential inhibition of 72- and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2. J Biol Chem. 1991 Jul 15;266(20):13070–13075. [PubMed] [Google Scholar]
  17. Imren S., Kohn D. B., Shimada H., Blavier L., DeClerck Y. A. Overexpression of tissue inhibitor of metalloproteinases-2 retroviral-mediated gene transfer in vivo inhibits tumor growth and invasion. Cancer Res. 1996 Jul 1;56(13):2891–2895. [PubMed] [Google Scholar]
  18. Jones S. E., Jomary C., Neal M. J. Expression of TIMP3 mRNA is elevated in retinas affected by simplex retinitis pigmentosa. FEBS Lett. 1994 Sep 26;352(2):171–174. doi: 10.1016/0014-5793(94)00951-1. [DOI] [PubMed] [Google Scholar]
  19. Katayose D., Wersto R., Cowan K. H., Seth P. Effects of a recombinant adenovirus expressing WAF1/Cip1 on cell growth, cell cycle, and apoptosis. Cell Growth Differ. 1995 Oct;6(10):1207–1212. [PubMed] [Google Scholar]
  20. Katayose D., Wersto R., Cowan K., Seth P. Consequences of p53 gene expression by adenovirus vector on cell cycle arrest and apoptosis in human aortic vascular smooth muscle cells. Biochem Biophys Res Commun. 1995 Oct 13;215(2):446–451. doi: 10.1006/bbrc.1995.2485. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Li Z., Li L., Zielke H. R., Cheng L., Xiao R., Crow M. T., Stetler-Stevenson W. G., Froehlich J., Lakatta E. G. Increased expression of 72-kd type IV collagenase (MMP-2) in human aortic atherosclerotic lesions. Am J Pathol. 1996 Jan;148(1):121–128. [PMC free article] [PubMed] [Google Scholar]
  25. Martin D. C., Rüther U., Sanchez-Sweatman O. H., Orr F. W., Khokha R. Inhibition of SV40 T antigen-induced hepatocellular carcinoma in TIMP-1 transgenic mice. Oncogene. 1996 Aug 1;13(3):569–576. [PubMed] [Google Scholar]
  26. McGrory W. J., Bautista D. S., Graham F. L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. doi: 10.1016/0042-6822(88)90302-9. [DOI] [PubMed] [Google Scholar]
  27. Montgomery A. M., Mueller B. M., Reisfeld R. A., Taylor S. M., DeClerck Y. A. Effect of tissue inhibitor of the matrix metalloproteinases-2 expression on the growth and spontaneous metastasis of a human melanoma cell line. Cancer Res. 1994 Oct 15;54(20):5467–5473. [PubMed] [Google Scholar]
  28. Murphy A. N., Unsworth E. J., Stetler-Stevenson W. G. Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation. J Cell Physiol. 1993 Nov;157(2):351–358. doi: 10.1002/jcp.1041570219. [DOI] [PubMed] [Google Scholar]
  29. Murphy G., Willenbrock F., Ward R. V., Cockett M. I., Eaton D., Docherty A. J. The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases. Biochem J. 1992 May 1;283(Pt 3):637–641. doi: 10.1042/bj2830637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pauly R. R., Passaniti A., Bilato C., Monticone R., Cheng L., Papadopoulos N., Gluzband Y. A., Smith L., Weinstein C., Lakatta E. G. Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ Res. 1994 Jul;75(1):41–54. doi: 10.1161/01.res.75.1.41. [DOI] [PubMed] [Google Scholar]
  31. Ray J. M., Stetler-Stevenson W. G. Gelatinase A activity directly modulates melanoma cell adhesion and spreading. EMBO J. 1995 Mar 1;14(5):908–917. doi: 10.1002/j.1460-2075.1995.tb07072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Silbiger S. M., Jacobsen V. L., Cupples R. L., Koski R. A. Cloning of cDNAs encoding human TIMP-3, a novel member of the tissue inhibitor of metalloproteinase family. Gene. 1994 Apr 20;141(2):293–297. doi: 10.1016/0378-1119(94)90588-6. [DOI] [PubMed] [Google Scholar]
  34. Southgate K. M., Davies M., Booth R. F., Newby A. C. Involvement of extracellular-matrix-degrading metalloproteinases in rabbit aortic smooth-muscle cell proliferation. Biochem J. 1992 Nov 15;288(Pt 1):93–99. doi: 10.1042/bj2880093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Southgate K. M., Fisher M., Banning A. P., Thurston V. J., Baker A. H., Fabunmi R. P., Groves P. H., Davies M., Newby A. C. Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries. Circ Res. 1996 Dec;79(6):1177–1187. doi: 10.1161/01.res.79.6.1177. [DOI] [PubMed] [Google Scholar]
  36. Stetler-Stevenson W. G., Brown P. D., Onisto M., Levy A. T., Liotta L. A. Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues. J Biol Chem. 1990 Aug 15;265(23):13933–13938. [PubMed] [Google Scholar]
  37. 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]
  38. Uría J. A., Ferrando A. A., Velasco G., Freije J. M., López-Otín C. Structure and expression in breast tumors of human TIMP-3, a new member of the metalloproteinase inhibitor family. Cancer Res. 1994 Apr 15;54(8):2091–2094. [PubMed] [Google Scholar]
  39. Walther S. E., Denhardt D. T. Directed mutagenesis reveals that two histidines in tissue inhibitor of metalloproteinase-1 are each essential for the suppression of cell migration, invasion, and tumorigenicity. Cell Growth Differ. 1996 Nov;7(11):1579–1588. [PubMed] [Google Scholar]
  40. Ward R. V., Atkinson S. J., Slocombe P. M., Docherty A. J., Reynolds J. J., Murphy G. Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes. Biochim Biophys Acta. 1991 Aug 30;1079(2):242–246. doi: 10.1016/0167-4838(91)90132-j. [DOI] [PubMed] [Google Scholar]
  41. Watanabe M., Takahashi Y., Ohta T., Mai M., Sasaki T., Seiki M. Inhibition of metastasis in human gastric cancer cells transfected with tissue inhibitor of metalloproteinase 1 gene in nude mice. Cancer. 1996 Apr 15;77(8 Suppl):1676–1680. doi: 10.1002/(SICI)1097-0142(19960415)77:8<1676::AID-CNCR38>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]
  42. Weber B. H., Vogt G., Pruett R. C., Stöhr H., Felbor U. Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy. Nat Genet. 1994 Dec;8(4):352–356. doi: 10.1038/ng1294-352. [DOI] [PubMed] [Google Scholar]
  43. Wick M., Bürger C., Brüsselbach S., Lucibello F. C., Müller R. A novel member of human tissue inhibitor of metalloproteinases (TIMP) gene family is regulated during G1 progression, mitogenic stimulation, differentiation, and senescence. J Biol Chem. 1994 Jul 22;269(29):18953–18960. [PubMed] [Google Scholar]
  44. Wilkinson G. W., Akrigg A. Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. Nucleic Acids Res. 1992 May 11;20(9):2233–2239. doi: 10.1093/nar/20.9.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Will H., Atkinson S. J., Butler G. S., Smith B., Murphy G. The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3. J Biol Chem. 1996 Jul 19;271(29):17119–17123. doi: 10.1074/jbc.271.29.17119. [DOI] [PubMed] [Google Scholar]
  46. Willenbrock F., Murphy G. Structure-function relationships in the tissue inhibitors of metalloproteinases. Am J Respir Crit Care Med. 1994 Dec;150(6 Pt 2):S165–S170. doi: 10.1164/ajrccm/150.6_Pt_2.S165. [DOI] [PubMed] [Google Scholar]
  47. Yang T. T., Hawkes S. P. Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10676–10680. doi: 10.1073/pnas.89.22.10676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zempo N., Kenagy R. D., Au Y. P., Bendeck M., Clowes M. M., Reidy M. A., Clowes A. W. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J Vasc Surg. 1994 Aug;20(2):209–217. doi: 10.1016/0741-5214(94)90008-6. [DOI] [PubMed] [Google Scholar]
  49. el-Deiry W. S., Harper J. W., O'Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994 Mar 1;54(5):1169–1174. [PubMed] [Google Scholar]

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