Phorbol Ester-induced Transcription of a Fibroblast Growth Factor-binding Protein Is Modulated by a Complex Interplay of Positive and Negative Regulatory Promoter Elements

Abstract

Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels 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 increased FGF-BP mRNA levels in a time-and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steadystate levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology 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 expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPAinducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element.

Downmodulation of bFGF-binding protein expression following restoration of p53 function

“…Consequently, tumor growth and angiogenesis in vivo are reduced. 63,66 The data presented here in our study show that Advmediated introduction of wtp53 into a human SCCHN cell line resulted in a time -dependent down -regulation of the expression of bFGF -BP. This downmodulation of bFGF -BP expression in vitro was evident by day 2.…”

Krüppel-like factor 5 promotes breast cell proliferation partially through upregulating the transcription of fibroblast growth factor binding protein 1

“…First, both KLF5 and FGF-BP were reported to be induced by TPA (Harris et al, 1998;Sun et al, 2001;Sur et al, 2002) and the MAPK pathway (Harris et al, 2001;Sun et al, 2001;Chanchevalap et al, 2006). In addition, microarray studies suggest that FGF-BP is a downstream gene of KLF5 in vitro and in vivo (Chen et al, 2006;Wan et al, 2008).…”

“…Both KLF5 and FGF-BP have been shown to be induced by phorbol ester (phorbol 12-myristate 13-acetate, TPA) (Harris et al, 1998;Sun et al, 2001). If FGF-BP is a KLF5 target gene, it is possible that KLF5 is required for TPA to induce FGF-BP.…”

“…To test this, we manually analysed the FGF-BP proximal promoter (Harris et al, 1998(Harris et al, , 2001Kagan et al, 2003) and found two potential KLF5 binding sites (Figure 4a). Then, we generated two luciferase reporter constructs by cloning the FGF-BP promoter (À217/ þ 61 and À128/ þ 61) into the PGL3-BASIC plasmid.…”

“…(a) The FGF-BP proximal promoter sequence (À217 to þ 61) contains two putative KLF5 binding sites as underlined. The transcription start site and other transcription factor binding sites are also indicated (Harris et al, 1998). (b) KLF5 activates the FGF-BP promoter through a GC-rich box.…”