Characterisation of the rat heparin-binding epidermal growth factor-like growth factor gene promoter

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene ex pression is strongly activated by a variety of extracellular stimuli, actin g through the Raf/MEK/MAP kinase pathway. To study the elements that respon d to this pathway, we have isolated and sequenced a fragment of the rat HB- EGF gene promoter. By transfection of a series of promoter/reporter constru cts into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, ga ve a fold-stimulation of activity no greater than that seen for the parenta l pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced lit tle or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB -EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5'-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two specie s approximately 260 bp upstream of the transcriptional start site. PCR anal ysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in th e HB-EGF gene. (C) 2000 Elsevier Science B.V. All rights reserved.