PHORBOL ESTER-INDUCED TRANSCRIPTION OF A FIBROBLAST GROWTH FACTOR-BINDING PROTEIN IS MODULATED BY A COMPLEX INTERPLAY OF POSITIVE AND NEGATIVE REGULATORY PROMOTER ELEMENTS

Earlier studies from our laboratory showed that a secreted binding pro tein for fibroblast growth factors (FGF-BP) is expressed at high level s in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF- BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA i ncreased FGF-BP mRNA levels in a time- and dose-dependent manner media ted via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transc ription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independen tly of de novo protein synthesis. We isolated the human FGF-BP promote r and determined by deletion analysis that TPA regulatory elements wer e all contained in the first 118 base pairs upstream of the transcript ion start site. Further mutational analysis revealed that full TPA ind uction required interplay between several regulatory elements with hom ology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites, In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expressi on. This region represses the extent of the FGF-BP promoter response t o TPA and contained sequences recognized by the family of E box helix- loop-helix transcription factors. Gel shift analysis showed specific a nd TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Fu rthermore, distinct, specific, and TPA-inducible binding to the imperf ect E box repressor element was also apparent. Overall, our data indic ate that TPA effects on FGF-BP gene transcription are tightly controll ed by a complex interplay of positive elements and a novel negative re gulatory element.