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Papers In Press, published online ahead of print January 4, 2002
J. Biol. Chem, 10.1074/jbc.M105682200
Submitted on June 20, 2001
Revised on December 21, 2001
Accepted on January 4, 2002
Department of General Surgery, Rush Presbyterian St. Luke's Medical Center, Chicago, IL 60612
Corresponding Author: xxu{at}rush.edu
Heparanase-1 (HPR1) is an endoglycosidase that specifically degrades the heparan sulfate chains of proteoglycan (HSPG), a component of blood vessel walls and the extracellular matrix (ECM). Recent studies demonstrated that HPR1 expression is increased in a variety of malignancies and may play a critical role in tumor metastases. The gene of HPR1 and its genomic structure has been recently cloned and characterized. To understand the mechanisms of HPR1 gene expression and regulation, we first mapped the transcription start site of the HPR1 gene and found that HPR1 mRNA was transcribed from the nucleotide position of 101-bp upstream of the ATG codon. A 3.5-kb promoter region of the HPR1 gene was cloned. Sequence analysis revealed that the TATA-less, GC-rich promoter of the HRP gene belongs to the family of housekeeping genes. This 3.5-kb promoter region exhibited strong promoter activity in two thyroid tumor cell lines. Truncational analysis of the HPR1 promoter identified a minimal 0.3-kb region that had a strong basal promoter activity. Truncational and mutational analysis of the HPR1 promoter revealed three Sp1 sites and four Ets-relevant elements (ERE) significantly contributing to basal HPR1 promoter activity. The binding to the Sp1 sites by Sp1 and to the ERE sites by GA-binding protein (GABP) was confirmed by electrophoretic mobility shift assay (EMSA), competition and supershift EMSA. Co-transfection of Sp- and GABP-deficient Drosophila SL-2 cells with the HPR1 promoter-driven luciferase construct plus the expression vector encoding the Sp1, Sp3, or GABP genes induced luciferase gene expression. Mutation or truncation of the Sp1 or ERE sites reduced luciferase expression in both SL-2 cells and thyroid tumor cell lines. Co-expression of GABPalpha+beta and Sp1 or Sp3 further increased luciferase reporter gene expression. Our results collectively suggest that Sp1 cooperates with GABP to regulate HPR1 promoter activity.
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