CpG methylation of the PAI-1 gene 5’-flanking region is inversely correlated with PAI-1 mRNA levels in human cell lines

Shan Gao; Sune Skeldal; Annelise Krogdahl; Jens Ahm Sørensen; Peter A. Andreasen

doi:10.1160/TH05-02-0114

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(03): 651-660
DOI: 10.1160/TH05-02-0114

Cellular Proteolysis and Oncology

Schattauer GmbH

CpG methylation of the PAI-1 gene 5’-flanking region is inversely correlated with PAI-1 mRNA levels in human cell lines

Shan Gao

¹Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark

,

Sune Skeldal

¹Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark

,

Annelise Krogdahl

²Departments of Pathology

,

Jens Ahm Sørensen

³Plastic Surgery, Odense University Hospital, Odense, Denmark

,

Peter A. Andreasen

¹Department of Molecular Biology, University of Aarhus, Aarhus C, Denmark

› Author Affiliations

Abstract

Summary

The physiological and pathophysiological functions of PAI-1 are related to its expression by specific cell types in normal and diseased tissues. We analysed the contribution of DNA methylation to the variation in PAI-1 mRNA levels in five cell lines. We found varying frequencies of methylation of 25 CpGs in the -805/+152 region of the PAI-1 gene in Bowes, MCF-7 and U937 cells, while little or no methylation was detected in Hep2 and HT-1080 cells. The methylation frequency was inversely correlated with PAI-1 mRNA level within its 20-fold range in Bowes, MCF-7,U937,and Hep2 cells, while the lack of methylation in both Hep2 and HT- 1080 cells suggested another mechanism behind the 150-fold higher level in HT-1080 cells than in Hep2 cells. However, all cell lines exhibited a high frequency of methylation of 10 CpGs in a CpG island at about -1800. Treatment with 5-aza-2‘-deoxycytidine led up to circa a 40-fold increase in the PAI-1 mRNA level and a strong decrease in the frequency of methylation in the -805/+152 region in Bowes, MCF-7 and U937. The histone deacetylase inhibitor trichostatin A induced a several fold increase of the PAI-1 mRNA level in cells with a high methylation frequency of the -805/+152 region. As compared with matched normal tissue, three samples of oral squamous cell carcinomas displayed decreased frequencies of methylation of the PAI-1 5' flanking region and increased levels of PAI-1 mRNA. These results for the first time implicate DNA methylation and histone acetylation in regulation of the PAI-1 gene, and indicate that without proper CpG islands in 5’-flanking region, trancription may be regulated by methylation of less dense CpGs in the 5’-flanking region rather than methylation of upstream CpG island.

Keywords

PAI-1 - CpG methylation - histone acetylation - cell-specific - oral carcinoma

Full Text

References

References
1 Wind T, Hansen M, Jensen JK. et al. The molecular basis for anti-proteolytic and non-proteolytic functions of plasminogen activator inhibitor type-1: roles of the reactive centre loop, the shutter region, the flexible joint region and the small serpin fragment.. Biol Chem 2002; 383: 21-36.
2 Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis.. Cell Mol Life Sci 2000; 57: 25-40.
3 Vaughan DE. Angiotensin and vascular fibrinolytic balance.. Am J Hypertens 2002; 15: 3S-8S.
4 Durand MK, Bodker JS, Christensen A. et al. Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis.. Thromb Haemost 2004; 91: 438-49.
5 Nagamine Y, Medcalf RL, Munoz-Canoves P. Transcriptional and posttranscriptional regulation of the plasminogen activator system.. Thromb Haemost 2005; 93: 661-75.
6 Alessi MC, Juhan-Vague I. Contribution of PAI-1 in cardiovascular pathology.. Arch Mal Coeur Vaiss 2004; 97: 673-8.
7 Offersen BV, Nielsen BS, Hoyer-Hansen G. et al. The myofibroblast is the predominant plasminogen activator inhibitor-1-expressing cell type in human breast carcinomas.. Am J Pathol 2003; 163: 1887-99.
8 Illemann M, Hansen U, Nielsen HJ. et al. Leadingedge myofibroblasts in human colon cancer express plasminogen activator inhibitor-1.. Am J Clin Pathol 2004; 122: 256-65.
9 Usher PA, Thomsen OF, Iversen P. et al. Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue.. Int J Cancer 2005; 113: 870-80.
10 Attwood JT, Yung RL, Richardson BC. DNA methylation and the regulation of gene transcription.. Cell Mol Life Sci 2002; 59: 241-57.
11 Futscher BW, Oshiro MM, Wozniak RJ. et al. Role for DNA methylation in the control of cell type specific maspin expression.. Nat Genet 2002; 31: 175-9.
12 Strathdee G. et al. Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene.. Carcinogenesis 2004; 25: 693-701.
13 Esteller M. Cancer epigenetics: DNA methylation and chromatin alterations in human cancer.. Adv Exp Med Biol 2003; 532: 39-49.
14 Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics.. Trends Genet 2000; 16: 168-74.
15 Shen L, Fang J, Qiu D. et al. Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma.. Hepatogastroenterology 1998; 45: 1753-9.
16 Hanada M. et al. bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia.. Blood 1993; 82: 1820-8.
17 Gupta A, Godwin AK, Vanderveer L. et al. Hypomethylation of the synuclein gamma gene CpG islandpromotes its aberrant expression in breast carcinoma and ovarian carcinoma.. Cancer Res 2003; 63: 664-73.
18 Wong WK, Chen K, Shih JC. Decreased methylation and transcription repressor Sp3 up-regulated human monoamine oxidase (MAO) B expression during Caco-2 differentiation.. J Biol Chem 2003; 278: 36227-35.
19 Chicoine E, Esteve PO, Robledo O. et al. Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.. Biochem Biophys Res Commun 2002; 297: 765-72.
20 Nakayama M, Wada M, Harada T. et al. Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1 gene in acute myeloid leukemias.. Blood 1998; 92: 4296-307.
21 De Smet C, De Backer O, Faraoni I. et al. The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.. Proc Natl Acad Sci U. S.A 1996; 93: 7149-53.
22 Pakneshan P, Xing RH, Rabbani SA. Methylation status of uPA promoter as a molecular mechanism regulating prostate cancer invasion and growth in vitro and in vivo.. FASEB J 2003; 17: 1081-8.
23 Xing RH, Rabbani SA. Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cells: role of DNA methylation.. Int J Cancer 1999; 81: 443-50.
24 Lund LR, Georg B, Nielsen LS. et al. Plasminogen activator inhibitor type 1: cell-specific and differentiation- induced expression and regulation in human cell lines, as determined by enzyme-linked immunosorbent assay.. Mol Cell Endocrinol 1988; 60: 43-53.
25 Worm J, Bartkova J, Kirkin AF. et al. Aberrant p27Kip1 promoter methylation in malignant melanoma.. Oncogene 2000; 19: 5111-5.
26 Gao S. et al. Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma.. Int J Cancer 2004; 109: 230-7.
27 Klinger KW. et al. Plasminogen activator inhibitor type 1 gene is located at region q21.3-q22 of chromosome 7 and genetically linked with cystic fibrosis.. Proc Natl Acad Sci U. S.A 1987; 84: 8548-52.
28 Irigoyen JP, Munoz-Canoves P, Montero L. et al. The plasminogen activator system: biology and regulation.. Cell Mol Life Sci 1999; 56: 104-32.
29 Riccio A, Lund LR, Sartorio R. et al. The regulatory region of the human plasminogen activator inhibitor type-1 (PAI-1) gene.. Nucleic Acids Res 1988; 16: 2805-24.
30 Cameron EE, Bachman KE, Myohanen S. et al. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.. Nat Genet 1999; 21: 103-7.
31 Gonzalez-Zulueta M, Bender CM, Yang AS. et al. Methylation of the 5’ CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing.. Cancer Res 1995; 55: 4531-5.
32 Guilleret I, Benhattar J. Unusual distribution of DNA methylation within the hTERT CpG island in tissues and cell lines.. Biochem Biophys Res Commun 2004; 325: 1037-43.
33 Bender CM. et al. Roles of cell division and gene transcription in the methylation of CpG islands.. Mol Cell Biol 1999; 19: 6690-8.
34 Nguyen C, Liang G, Nguyen TT. et al. Susceptibility of nonpromoter CpG islands to de novo methylation in normal and neoplastic cells.. J Natl Cancer Inst 2001; 93: 1465-72.
35 Makar KW, Perez-Melgosa M, Shnyreva M. et al. Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells.. Nat Immunol 2003; 4: 1183-90.
36 Pogribny IP, Pogribna M, Christman JK. et al. Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis.. Cancer Res 2000; 60: 588-94.
37 Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics.. Science 2001; 293: 1068-70.
38 Keeton MR, Curriden SA, van Zonneveld AJ. et al. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta.. J Biol Chem 1991; 266: 23048-52.
39 Rice JC, Allis CD. Histone methylation versus histone acetylation: new insights into epigenetic regulation.. Curr Opin Cell Biol 2001; 13: 263-73.
40 Nielsen BS, Sehested M, Timshel S. et al. Messenger RNA for urokinase plasminogen activator is expressed in myofibroblasts adjacent to cancer cells in human breast cancer.. Lab Invest 1996; 74: 168-77.
41 Nozaki S, Endo Y, Kawashiri S. et al. Immunohistochemical localization of a urokinase-type plasminogen activator system in squamous cell carcinoma of the oral cavity: association with mode of invasion and lymph node metastasis.. Oral Oncol 1998; 34: 58-62.
42 Hou B, Eren M, Painter CA. et al. Tumor necrosis factor alpha activates the human plasminogen activator inhibitor-1 gene through a distal nuclear factor kappaB site.. J Biol Chem 2004; 279: 18127-36.
43 Fink T, Kazlauskas A, Poellinger L. et al. Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor. Blood 2002; 99: 2077-83.
44 Eriksson P, Nilsson L, Karpe F. et al. Very-low-density lipoprotein response element in the promoter region of the human plasminogen activator inhibitor-1 gene implicated in the impaired fibrinolysis of hypertriglyceridemia.. Arterioscler Thromb Vasc Biol 1998; 18: 20-6.
45 Dennler S, Itoh S, Vivien D. et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.. EMBO J 1998; 17: 3091-100.