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.
Highlights
We recently reported the identification of the serum inducible zinc finger transcription factor human early growth response gene 1 (EGR1) [41], as a key regulator of inducible HPSE transcription in T lymphocytes [42]
HPSE function has been implicated in tumorigenesis for over two decades, and the recent cloning of the HPSE gene has enabled experimental confirmation that it does play a key role in the metastasis of solid tumors as well as mediating tumor angiogenesis both directly by promoting endothelial cell migration and indirectly via release of proangiogenic molecules from the extracellular matrix (ECM) [27,28,29]
We have previously investigated regulatory mechanisms responsible for the normal induction of HPSE in T lymphocytes, and found that HPSE inducible expression in a Jurkat T cell model was regulated by the transcription factor EGR1 [42]
Summary
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. 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, 5Ј-TGTCCCCTGCAGCTCCAGC-3Ј and HEGR1B, 5Ј-ATAGACCTTCCACTCCAGTAG-3Ј. This fragment was cloned into pCR3.1 (Invitrogen) as per the manufacturer’s instructions. 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 [42]. Western blotting was used to detect HPSE in cell lysates using a rabbit polyclonal antibody specific for both 50- and 65-kDa human HPSE (generous gift of Dr Craig Freeman, The John Curtin School of Medical Research, Canberra) following the protocol described previously [31]. Chemiluminescence was detected using ECL Western blotting detection Reagents (Amersham Biosciences)
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