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• W2140691431 abstract "Heparanase is an endoglycosidase that degrades heparan sulfate chains of heparan sulfate proteoglycans, a key component of extracellular matrix and basement membranes. Studies using heparanase inhibitors and gene silencing have provided evidence to support an important role for heparanase in tumor metastasis and angiogenesis. The expression of heparanase is normally very tightly controlled, however, it is commonly deregulated in tumor cells, which express elevated heparanase activity that correlates with high levels of heparanase mRNA. We recently identified the transcription factor early growth response gene 1, EGR1, as a key regulator of inducible heparanase transcription in T cells. In this study using chromatin immunoprecipitation, we demonstrate for the first time that EGR1 binds to the heparanase gene promoter in vivo. The important question of the role of EGR1 in regulating heparanase transcription in tumor cells was then assessed. Studies were carried out in four epithelial tumor lines of different tissue origin. Functional dissection of the heparanase promoter identified a 280-bp region that was critical for transcription of the heparanase gene. Transactivation studies using an EGR1 expression vector co-transfected with a reporter construct containing the 280-bp region showed EGR1-activated heparanase promoter activity in a dose-dependent manner in prostate or breast adenocarcinoma and colon carcinoma cell lines. In contrast, overexpression of EGR1 resulted in a dose-dependent repression of promoter activity in melanoma cells. Using site-directed mutagenesis the 280-bp region was found to contain two functional EGR1 sites and electrophoretic mobility shift assays showed binding of EGR1 to both of these sites upon activation of tumor cells. Furthermore, the heparanase promoter region containing the EGR1 sites was also inducible in tumor cells and induction corresponded to HPSE expression levels. These studies show that EGR1 regulates heparanase transcription in tumor cells and importantly, can have a repressive or activating role depending on the tumor type. Heparanase is an endoglycosidase that degrades heparan sulfate chains of heparan sulfate proteoglycans, a key component of extracellular matrix and basement membranes. Studies using heparanase inhibitors and gene silencing have provided evidence to support an important role for heparanase in tumor metastasis and angiogenesis. The expression of heparanase is normally very tightly controlled, however, it is commonly deregulated in tumor cells, which express elevated heparanase activity that correlates with high levels of heparanase mRNA. We recently identified the transcription factor early growth response gene 1, EGR1, as a key regulator of inducible heparanase transcription in T cells. In this study using chromatin immunoprecipitation, we demonstrate for the first time that EGR1 binds to the heparanase gene promoter in vivo. The important question of the role of EGR1 in regulating heparanase transcription in tumor cells was then assessed. Studies were carried out in four epithelial tumor lines of different tissue origin. Functional dissection of the heparanase promoter identified a 280-bp region that was critical for transcription of the heparanase gene. Transactivation studies using an EGR1 expression vector co-transfected with a reporter construct containing the 280-bp region showed EGR1-activated heparanase promoter activity in a dose-dependent manner in prostate or breast adenocarcinoma and colon carcinoma cell lines. In contrast, overexpression of EGR1 resulted in a dose-dependent repression of promoter activity in melanoma cells. Using site-directed mutagenesis the 280-bp region was found to contain two functional EGR1 sites and electrophoretic mobility shift assays showed binding of EGR1 to both of these sites upon activation of tumor cells. Furthermore, the heparanase promoter region containing the EGR1 sites was also inducible in tumor cells and induction corresponded to HPSE expression levels. These studies show that EGR1 regulates heparanase transcription in tumor cells and importantly, can have a repressive or activating role depending on the tumor type. Understanding the molecular basis of tumor metastasis and angiogenesis remains a key focus in cancer research and is critical for the development of novel interventional approaches in the treatment of neoplastic pathologies. The extracellular matrix (ECM) 3The abbreviations used are: ECMextracellular matrixChIPchromatin immunoprecipitationHPSEheparanaseEGR1early growth response gene 1PMAphorbol 12-myristate 13-acetateRTreverse transcriptase. is an important structure in these processes, in particular the specialized form known as basement membranes that surround vessels and provides a physical barrier to the migration of cells. An essential structural component of the ECM and also cell surfaces are heparan sulfate proteoglycans. These are composed of a protein core covalently linked to complex sulfated glycosaminoglycan, heparan sulfate side chains (1Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar, 2Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2323) Google Scholar, 3Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1678) Google Scholar), that interact with other components of the ECM, such as fibronectin, collagen, and laminin, to provide matrix assembly and stability. The main mechanism of heparan sulfate cleavage is by the β-d-endoglucuronidase, heparanase (HPSE) (4Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 5Vlodavsky I. Friedmann Y. J. Clin. Investig. 2001; 108: 341-347Crossref PubMed Scopus (548) Google Scholar). The cleavage of heparan sulfate chains by heparanase expressing cells such as metastatic tumor cells, proliferating endothelial cells, and activated leukocytes facilitates the degradation of the ECM promoting cell invasion associated with tumor metastasis, angiogenesis, and inflammation (6Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 7Ratner S. Invasion Metastasis. 1992; 12: 82-100PubMed Google Scholar). In addition, heparan sulfate chains in the ECM specifically bind many proteins such as growth factors and cytokines (2Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2323) Google Scholar) that upon cleavage by HPSE, modulate the environment of the ECM to facilitate angiogenic responses required for wound healing and tumor angiogenesis (8Vlodavsky I. Elkin M. Pappo O. Aingorn H. Atzmon R. Ishai-Michaeli R. Aviv A. Pecker I. Friedmann Y. Isr. Med. Assoc. J. 2000; 2: 37-45PubMed Google Scholar). The cloning and characterization of HPSE (6Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 9Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 10Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar, 11Dempsey L.A. Plummer T.B. Coombes S.L. Platt J.L. Glycobiology. 2000; 10: 467-475Crossref PubMed Scopus (85) Google Scholar) has suggested that this gene encodes for the dominant heparan sulfate-degrading enzyme in mammalian tissues. Consequently, HPSE has become a very attractive drug target for new anti-metastatic and anti-angiogenic therapies. extracellular matrix chromatin immunoprecipitation heparanase early growth response gene 1 phorbol 12-myristate 13-acetate reverse transcriptase. Human HPSE is expressed as a 543-amino acid, 65-kDa proenzyme (6Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar, 10Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar) that is proteolytically processed to produce an active enzyme composed of 8- and 50-kDa polypeptide chains that form a heterodimer (12Fairbanks M.B. Mildner A.M. Leone J.W. Cavey G.S. Mathews W.R. Drong R.F. Slightom J.L. Bienkowski M.J. Smith C.W. Bannow C.A. Heinrikson R.L. J. Biol. Chem. 1999; 274: 29587-29590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 13McKenzie E. Young K. Hircock M. Bennett J. Bhaman M. Felix R. Turner P. Stamps A. McMillan D. Saville G. Ng S. Mason S. Snell D. Schofield D. Gong H. Townsend R. Gallagher J. Page M. Parekh R. Stubberfield C. Biochem. J. 2003; 373: 423-435Crossref PubMed Scopus (104) Google Scholar, 14Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). Northern blot analysis has identified two major transcripts in human, a 1.7- and 5-kb mRNA (10Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (486) Google Scholar, 15Dong J. Kukula A.K. Toyoshima M. Nakajima M. Gene (Amst.). 2000; 253: 171-178Crossref PubMed Scopus (75) Google Scholar), which encode for the same protein product. HPSE expression under normal physiological conditions is restricted to activated leukocytes, endothelial cells, and smooth muscle cells (16Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 17Godder K. Vlodavsky I. Eldor A. Weksler B.B. Haimovitz-Freidman A. Fuks Z. J. Cell. Physiol. 1991; 148: 274-280Crossref PubMed Scopus (34) Google Scholar), as well as cytotrophoblasts, keratinocytes, and platelets (11Dempsey L.A. Plummer T.B. Coombes S.L. Platt J.L. Glycobiology. 2000; 10: 467-475Crossref PubMed Scopus (85) Google Scholar, 16Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 18Goshen R. Hochberg A.A. Korner G. Levy E. Ishai-Michaeli R. Elkin M. de Groot N. Vlodavsky I. Mol. Hum. Reprod. 1996; 2: 679-684Crossref PubMed Scopus (56) Google Scholar, 19Bernard D. Mehul B. Delattre C. Simonetti L. Thomas-Collignon A. Schmidt R. J. Invest. Dermatol. 2001; 117: 1266-1273Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, HPSE expression is also up-regulated by many tumor cells. Numerous studies of clinical tumor samples have established that HPSE is highly expressed in tumors at both the protein and mRNA levels (20Gohji K. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Katsuoka Y. Nakajima M. J. Urol. 2001; 166: 1286-1290Crossref PubMed Scopus (100) Google Scholar, 21Simizu S. Ishida K. Wierzba M.K. Sato T.A. Osada H. Cancer Lett. 2003; 193: 83-89Crossref PubMed Scopus (43) Google Scholar, 22Koliopanos A. Friess H. Kleeff J. Shi X. Liao Q. Pecker I. Vlodavsky I. Zimmermann A. Buchler M.W. Cancer Res. 2001; 61: 4655-4659PubMed Google Scholar, 23Friedmann Y. Vlodavsky I. Aingorn H. Aviv A. Peretz T. Pecker I. Pappo O. Am. J. Pathol. 2000; 157: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and its expression is correlated with disease (22Koliopanos A. Friess H. Kleeff J. Shi X. Liao Q. Pecker I. Vlodavsky I. Zimmermann A. Buchler M.W. Cancer Res. 2001; 61: 4655-4659PubMed Google Scholar, 24Sato T. Yamaguchi A. Goi T. Hirono Y. Takeuchi K. Katayama K. Matsukawa S. J. Surg. Oncol. 2004; 87: 174-181Crossref PubMed Scopus (74) Google Scholar). Furthermore, studies using HPSE inhibitors (25Parish C.R. Freeman C. Brown K.J. Francis D.J. Cowden W.B. Cancer Res. 1999; 59: 3433-3441PubMed Google Scholar, 26Vlodavsky I. Mohsen M. Lider O. Svahn C.M. Ekre H.P. Vigoda M. Ishai-Michaeli R. Peretz T. Invasion Metastasis. 1994; 14: 290-302PubMed Google Scholar) or HPSE gene silencing approaches (27Zhang Y.L. Fu Z.R. Zhang J. Wang Y.H. Shen Q. Zhonghua Yi Xue Za Zhi. 2003; 83: 204-207PubMed Google Scholar, 28Uno F. Fujiwara T. Takata Y. Ohtani S. Katsuda K. Takaoka M. Ohkawa T. Naomoto Y. Nakajima M. Tanaka N. Cancer Res. 2001; 61: 7855-7860PubMed Google Scholar, 29Edovitsky E. Elkin M. Zcharia E. Peretz T. Vlodavsky I. J. Natl. Cancer Inst. 2004; 96: 1219-1230Crossref PubMed Scopus (227) Google Scholar) have confirmed the important in vivo role of HPSE in the processes of tumor metastasis, angiogenesis, and growth. Recent studies are attempting to identify the mechanisms by which HPSE switches from being a normally tightly controlled gene to one that is deregulated in tumor cells. Whereas advances have been made into understanding the processing of the proenzyme (14Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar, 30Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 31Hulett M.D. Hornby J.R. Ohms S.J. Zuegg J. Freeman C. Gready J.E. Parish C.R. Biochemistry. 2000; 39: 15659-15667Crossref PubMed Scopus (143) Google Scholar, 32Abboud-Jarrous G. Aingorn H. Rangini-Guetta Z. Atzmon R. Elgavish S. Peretz T. Vlodavsky I. J. Biol. Chem. 2005; 280: 13568-13575Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and the environmental factors that influence its activity (33Ihrcke N.S. Parker W. Reissner K.J. Platt J.L. J. Cell. Physiol. 1998; 175: 255-267Crossref PubMed Scopus (76) Google Scholar, 34Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2002; 115: 2179-2187Crossref PubMed Google Scholar, 35Han J. Woytowich A. Mandal A. Hiebert L. Cardiovasc. Pathol. 2004; 13: 85Crossref PubMed Google Scholar), our knowledge on the mechanisms of the complex regulatory processes of HPSE gene transcription remains limited. Two studies have highlighted the importance of several ETS response elements in the promoter, although the emphasis in each study were of different sites and the studies were limited to a breast carcinoma line (36Lu W.C. Liu Y.N. Kang B.B. Chen J.H. Oncogene. 2003; 22: 919-923Crossref PubMed Scopus (56) Google Scholar) and thyroid carcinoma lines (37Jiang P. Kumar A. Parrillo J.E. Dempsey L.A. Platt J.L. Prinz R.A. Xu X. J. Biol. Chem. 2002; 277: 8989-8998Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Jiang et al. (37Jiang P. Kumar A. Parrillo J.E. Dempsey L.A. Platt J.L. Prinz R.A. Xu X. J. Biol. Chem. 2002; 277: 8989-8998Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) also showed there were three functional Sp1 sites in the promoter that acted cooperatively with the ETS family member, GABP. At the endogenous gene level, it has been shown that methylation of a CpG region in the HPSE promoter can contribute to the silencing of the gene (21Simizu S. Ishida K. Wierzba M.K. Sato T.A. Osada H. Cancer Lett. 2003; 193: 83-89Crossref PubMed Scopus (43) Google Scholar, 38Shteper P.J. Zcharia E. Ashhab Y. Peretz T. Vlodavsky I. Ben-Yehuda D. Oncogene. 2003; 22: 7737-7749Crossref PubMed Scopus (101) Google Scholar, 39Ogishima T. Shiina H. Breault J.E. Tabatabai L. Bassett W.W. Enokida H. Li L.C. Kawakami T. Urakami S. Ribeiro-Filho L.A. Terashima M. Fujime M. Igawa M. Dahiya R. Clin. Cancer Res. 2005; 11: 1028-1036PubMed Google Scholar) and more recently, the expression of a dominant negative form of cAMP-response element-binding protein was shown to decrease HPSE mRNA levels in melanoma cells (40Aucoin R. Reiland J. Roy M. Marchetti D. J. Cell. Biochem. 2004; 93: 215Crossref PubMed Scopus (11) Google Scholar). We recently reported the identification of the serum inducible zinc finger transcription factor human early growth response gene 1 (EGR1) (41Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar), as a key regulator of inducible HPSE transcription in T lymphocytes (42de Mestre A.M. Khachigian L.M. Santiago F.S. Staykova M.A. Hulett M.D. J. Biol. Chem. 2003; 278: 50377-50385Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These current studies address the important question as to whether EGR1 may also be playing a key role in the deregulated expression of HPSE in human tumor cells. EGR1 is a member of the early growth response family that also includes, EGR2, EGR3, EGR4, and WT-1. This family of transcription factors share the ability to bind to GC-rich recognition motifs in DNA (41Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar). EGR1 is a nuclear phospho-protein that is rapidly induced in response to a variety of extracellular and environmental signals (including growth factors, cytokines, vascular injury, and hypoxia) (41Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 43Khachigian L.M. Anderson K.R. Halnon N.J. Gimbrone Jr., M.A. Resnick N. Collins T. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2280-2286Crossref PubMed Scopus (174) Google Scholar, 44Yan S.F. Lu J. Zou Y.S. Soh-Won J. Cohen D.M. Buttrick P.M. Cooper D.R. Steinberg S.F. Mackman N. Pinsky D.J. Stern D.M. J. Biol. Chem. 1999; 274: 15030-15040Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) where it binds to the promoters of a range of genes (45Khachigian L.M. Collins T. J. Mol. Med. 1998; 76: 613-616Crossref PubMed Scopus (97) Google Scholar) to mediate responses such as wound healing and neo-vascularization, and has been strongly associated with vascular proliferative disorders (46Lowe H.C. Fahmy R.G. Kavurma M.M. Baker A. Chesterman C.N. Khachigian L.M. Circ. Res. 2001; 89: 670-677Crossref PubMed Scopus (106) Google Scholar, 47Fahmy R.G. Dass C.R. Sun L.Q. Chesterman C.N. Khachigian L.M. Nat. Med. 2003; 9: 1026-1032Crossref PubMed Scopus (301) Google Scholar). EGR1 expression has been shown to be variable between tumor cells of different tissue origin (48Huang R.P. Fan Y. de Belle I. Niemeyer C. Gottardis M.M. Mercola D. Adamson E.D. Int. J. Cancer. 1997; 72: 102-109Crossref PubMed Scopus (218) Google Scholar, 49Eid M.A. Kumar M.V. Iczkowski K.A. Bostwick D.G. Tindall D.J. Cancer Res. 1998; 58: 2461-2468PubMed Google Scholar, 50Kobayashi D. Yamada M. Kamagata C. Kaneko R. Tsuji N. Nakamura M. Yagihashi A. Watanabe N. Anticancer Res. 2002; 22: 3963-3970PubMed Google Scholar). The well characterized neo-vascularization promoting function of EGR1 in tissue injury suggests it would play an important role in tumor growth and tumor angiogenesis. Indeed, studies using knockdown strategies have confirmed a central in vivo role for EGR1 in tumor angiogenesis, growth, and metastasis in breast adenocarcinoma (47Fahmy R.G. Dass C.R. Sun L.Q. Chesterman C.N. Khachigian L.M. Nat. Med. 2003; 9: 1026-1032Crossref PubMed Scopus (301) Google Scholar, 51Mitchell A. Dass C.R. Sun L.Q. Khachigian L.M. Nucleic Acids Res. 2004; 32: 3065-3069Crossref PubMed Scopus (106) Google Scholar) and tumor progression in prostate adenocarcinomas (52Abdulkadir S.A. Qu Z. Garabedian E. Song S.K. Peters T.J. Svaren J. Carbone J.M. Naughton C.K. Catalona W.J. Ackerman J.J. Gordon J.I. Humphrey P.A. Milbrandt J. Nat. Med. 2001; 7: 101-107Crossref PubMed Scopus (155) Google Scholar, 53Baron V. Duss S. Rhim J. Mercola D. Ann. N. Y. Acad. Sci. 2003; 1002: 197-216Crossref PubMed Scopus (51) Google Scholar). A recent study of human prostate cancer samples has shown a correlation between EGR1 expression and HPSE mRNA expression (39Ogishima T. Shiina H. Breault J.E. Tabatabai L. Bassett W.W. Enokida H. Li L.C. Kawakami T. Urakami S. Ribeiro-Filho L.A. Terashima M. Fujime M. Igawa M. Dahiya R. Clin. Cancer Res. 2005; 11: 1028-1036PubMed Google Scholar), further supporting a role for EGR1 in regulating HPSE transcription in tumor cells. In this article we have functionally dissected the human HPSE promoter and identified EGR1 as critical in binding to two elements in the HPSE promoter and regulating transcription in tumor cells. Interestingly, EGR1 was found to have differential regulatory effects on HPSE transcription depending on the tumor type. These findings represent an important advance into understanding the mechanisms that control transcription of the HPSE gene in tumor cells. Cell Culture, Transfections, and Luciferase Assays—MCF7 human breast carcinoma cells were from American Type Culture Collection (ATCC, Manassas, VA), PC-3 human prostate adenocarcinoma cells (ATCC) MM170 human melanoma cells, COLO397 human colon carcinoma cells, and B16-F1 mouse melanoma cells were cultured in RPMI medium (Invitrogen) supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO2 and 37 °C. Transient transfections of MCF7, PC-3, COLO397, and B16-F10 cells were performed using Lipofectin (Invitrogen) as per the manufacturer's instructions. Briefly for the luciferase reporter experiments, cells at 70–80% confluence were transfected with luciferase reporter constructs and a Renilla luciferase construct, pRLTK (Promega), as an internal control. Transient transfection of MM170 melanoma cells were performed using Bio-Rad Gene Pulser II (Bio-Rad) at 270 volts and 975 capacitance with 5 μg of reporter construct and 1 μg of pRLTK being transfected per 4–5 × 106 cells. For the EGR1 overexpression experiments, each of the tumor cell lines were transfected as described above, with a constant amount of total DNA used, i.e. when decreasing amounts of pCR3.1-EGR-1 or pCB6-Egr1 were transfected, the balance was made up by co-transfection with the backbone constructs, pCR3.1 or pCB6+. Each of the tumor cell lines were transfected at similar efficiencies. After transfection, cells were rested for 24 h and then assayed for luciferase activity using a Dual-Glo™ luciferase assay system (Promega). Plates were read on a Reporter Microplate Luminometer (Turner Biosystems, Sunnyvale, CA). In activation assays, cells were rested for 24 h and then stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) for 16 h then assayed for luciferase activity as described above. Overexpression studies and mutagenesis effects were analyzed for statistical significance by using a two-tailed Student's t test. Plasmid Constructs—Luciferase reporter constructs pXP-1/1300bp, pXP-1/520bp, pXP-1/280bp, pXP-1/120bp, pXP-1/280bpMUT, and pCB6-Egr1 have been previously described (42de Mestre A.M. Khachigian L.M. Santiago F.S. Staykova M.A. Hulett M.D. J. Biol. Chem. 2003; 278: 50377-50385Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The pXP-1/280bpMUT2 construct was generated by splice overlap extension PCR by mutating the core sequence of the second putative EGR1 binding site, TGGG, to GTTT using the pXP-1/280bp construct as a template. Briefly two PCR were performed to generate overlapping 5′ and 3′ fragments encoding each mutation, which were then spliced together in a third PCR. The oligonucleotide primers used to generate these fragments were MTWT-1 and HHP1 or MTWT-2 and HHP3. The sequences of oligonucleotide primers MTWT-1, MTWT-2, HHP1, and HHP3 were 5′-AAGAGGAGGTTTAGGGATGGAGGGC-3′, 5′-TCCATCCCTAAACCTCCTCTTCTG-3′, 5′-TTCGAGCGCAGCAGCATC-3′ and 5′-GTAAGTGAACGTGACC-3, respectively. A 478-bp fragment of the mouse HPSE gene promoter was amplified by PCR from genomic DNA isolated from C57BL/6 mice, using oligonucleotide primers, MHP1 and MHP6. The amplified fragment was cloned into the BglII/KpnI site in pXP-1, to generate the vector pXP-1/0.5kb. The sequences of MHP1 and MHP6 were 5′-CAGCATCCCACCTGGCTG-3′ and 5′-TAAGGCAGAAGGGAGTC-3′, respectively. The pXP-1/0.5kbMUT1 construct was generated using the QuikChange® 11 XL Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) as described by the manufacturer, using pXP-1/0.5kb as a template. The core binding motif (underlined) of the putative Egr1 site, AGGGGTGGGAGG, was mutated to TGTT. The sequences of the oligonucleotide primers used to generate this mutation were: MEGRM1F, 5′-GGACTCCCGGGAGGGTGTTGAGGGATGGAGCGCTG-3′ and MEGRM1R, 5′-CAGCGCTCCATCCCTCAACACCCTCCCGGGAGTCC-3′. The EGR-1 expression vector, pCR3.1-EGR1, was generated using PCR to amplify a 1859-bp cDNA encoding for human EGR1 with oligonucleotides: HEGR1A, 5′-TGTCCCCTGCAGCTCCAGC-3′ and HEGR1B, 5′-ATAGACCTTCCACTCCAGTAG-3′. This fragment was cloned into pCR3.1 (Invitrogen) as per the manufacturer's instructions. The nucleotide sequence integrity of all clones was confirmed by automated sequencing (Biomolecular Resource Facility, The John Curtin School of Medical Research, Canberra, Australia) using an ABI 3730 Analyzer (Applied Biosystems, Foster City, CA) following the manufacturer's protocol. RNA Extraction and Real Time Quantitative RT-PCR—Total RNA was extracted from tumor cells at 80% confluence, using TriReagent® (Molecular Research Center Inc., Cincinnati, OH) as described by the manufacturer. cDNA synthesis was performed on 1 μg of total RNA using Superscript™ 111 Reverse Transcriptase as per the manufacturer's instructions (Invitrogen). SYBR Green (Qiagen GmbH, Hilden, Germany) real time PCR for amplification of heparanase, EGR1, or the housekeeper gene ubiquitin-conjugating enzyme, E2D 2, were performed using a ABI PRISM 7700 sequence detector (PerkinElmer Life Sciences) as previously described (42de Mestre A.M. Khachigian L.M. Santiago F.S. Staykova M.A. Hulett M.D. J. Biol. Chem. 2003; 278: 50377-50385Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A dissociation curve was performed after each experiment to confirm a single product was amplified. A standard curve was generated for each gene using known copy numbers of a plasmid containing the cDNA specific to the gene. Chromatin Immunoprecipitation (ChIP) Assay—ChIP analysis was performed on 5 × 106 Jurkat T cells either non-stimulated or following stimulation with PMA (20 ng/ml) and calcium ionophore (Sigma) (1 μm) for 4 or 12 h following a protocol previously described with some modifications (54Litt M.D. Simpson M. Recillas-Targa F. Prioleau M.N. Felsenfeld G. EMBO J. 2001; 20: 2224-2235Crossref PubMed Scopus (323) Google Scholar, 55Chen X. Wang J. Woltring D. Gerondakis S. Shannon M.F. Mol. Cell. Biol. 2005; 25: 3209-3219Crossref PubMed Scopus (89) Google Scholar). In brief, cells were harvested and cross-linked with 1% formaldehyde for 10 min, and the reaction was terminated by the addition of 0.25 m glycine. Cells were washed four times in ice-cold phosphate-buffered saline, resuspended in ChIP SDS lysis buffer (Upstate Biotechnology, Charlottesville, VA) in the presence of Complete protease inhibitors (Roche Diagnostics), and sonicated to shear chromatin using a Cole Palmer Ultrasonic processor (Cole Palmer, Vernon Hills, IL). The sonicated DNA fragments were in the range of 100 to 1000 bp. The samples were pre-cleared with 60 μl of salmon sperm DNA-protein A-agarose (Upstate Biotechnology) and subsequently incubated with either 4 μg of anti-EGR1 antibody Egr1X-588 (Santa Cruz Biotechnology) or without antibody as a control, overnight with rotation at 4 °C. Immunocomplexes were recovered with salmon sperm DNA-protein A-agarose (Upstate Biotechnology), washed extensively as described previously (55Chen X. Wang J. Woltring D. Gerondakis S. Shannon M.F. Mol. Cell. Biol. 2005; 25: 3209-3219Crossref PubMed Scopus (89) Google Scholar), and eluted with ChIP elution buffer (Upstate Biotechnology). Following the reversal of cross-links at 65 °C overnight, samples were extracted with phenol/chloroform and resuspended in Milli Q water for real time PCR analysis. ChIP-purified DNA was screened for HPSE and CD69 proximal promoter fragments or –1-kb HPSE promoter fragments by real time PCR analysis as described previously (54Litt M.D. Simpson M. Recillas-Targa F. Prioleau M.N. Felsenfeld G. EMBO J. 2001; 20: 2224-2235Crossref PubMed Scopus (323) Google Scholar, 55Chen X. Wang J. Woltring D. Gerondakis S. Shannon M.F. Mol. Cell. Biol. 2005; 25: 3209-3219Crossref PubMed Scopus (89) Google Scholar). The sequence for the oligonucleotide primers were: HPSE set A (proximal HPSE promoter) sense, 5′-TTCGTAAGTGAACGTCACCG-3′ and antisense, 5′-CTTCTGCATCCCTCCCACT-3′; HPSE set B (–1 kb HPSE promoter) sense, 5′-TTCTGACACTTCACATCCCG-3′ and antisense, 5′-AACCTGCCAAGTGCATACT-3′; and CD69 (proximal CD69 promoter) sense, 5′-AATCCCACTTTCCTCCTGCT-3′ and antisense, 5′-GCCGCCTACTTGCTTGACTA-3′ The amount of precipitated target sequence was calculated by normalization with the total input DNA after subtraction of the no antibody background as described previously (54Litt M.D. Simpson M. Recillas-Targa F. Prioleau M.N. Felsenfeld G. EMBO J. 2001; 20: 2224-2235Crossref PubMed Scopus (323) Google Scholar, 55Chen X. Wang J. Woltring D. Gerondakis S. Shannon M.F. Mol. Cell. Biol. 2005; 25: 3209-3219Crossref PubMed Scopus (89) Google Scholar). Nuclear Extraction and Electrophoretic Mobility Shift Assay—PC-3 or MCF7 cells at 80% confluence were harvested and washed in 10 ml of ice-cold phosphate-buffered saline either non-stimulated or after activation with PMA for 1.5 h. Nuclear extraction was then performed as previously described (56Day F.L. Rafty L.A. Chesterman C.N. Khachigian L.M. J. Biol. Chem. 1999; 274: 23726-23733Abstract Full Text Full Te" @default.
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