Serum induction of the fibroblast growth factor-binding protein (FGF-BP) is mediated through ERK and p38 MAP kinase activation and C/EBP-regulated transcription.

The fibroblast growth factor-binding protein (FGF-BP) modulates FGF activity through binding and release from the extracellular matrix. Consequently, the expression of FGF-BP in certain tumor types is a rate-limiting regulator of FGF-mediated angiogenesis. FGF-BP is upregulated in squamous cell carcinoma by treatment with mitogens such as EGF or TPA. In this study, we investigated the regulation of FGF-BP gene expression by serum. Treatment of serum-starved ME-180 cells with fetal bovine serum (FBS) resulted in a rapid increase in steady-state levels of FGF-BP mRNA and in the rate of FGF-BP gene transcription. Serum induction of FGF-BP mRNA was not mediated through EGF receptor activation but was dependent on PKC, as well as ERK kinase (MEK) and p38 MAP kinase activation. Promoter analysis showed that C/EBP is the main promoter element required for the serum response. Unlike EGF-activation of FGF-BP, transcriptional induction by serum is not significantly regulated through the AP-1 or E-box sites in the promoter. These results illustrate differences between the mechanism of induction in response to serum and EGF.

Mitogen-induced expression of the fibroblast growth factor-binding protein is transcriptionally repressed through a non-canonical E-box element.

The fibroblast growth factor-binding protein (FGF-BP) stimulates FGF-2-mediated angiogenesis and is thought to play an important role in the progression of squamous cell, colon, and breast carcinomas. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induction of the FGF-BP gene occurs through transcriptional mechanisms involving Sp1, AP-1, and CCAATT/enhancer-binding protein sites in the proximal FGF-BP gene promoter. The level of TPA induction, however, is limited due to the presence of a repressor element that shows similarity to a non-canonical E-box (AACGTG). Mutation or deletion of the repressor element led to enhanced induction by TPA or epidermal growth factor in cervical squamous cell and breast carcinoma cell lines. Repression was dependent on the adjacent AP-1 site, without discernible alteration in the binding affinity or composition of AP-1. We investigated the following two possible mechanisms for E-box-mediated repression: 1) CpG methylation of the core of the E-box element, and 2) binding of a distinct protein complex to this site. Point mutation of the CpG methylation site in the E-box showed loss of repressor activity. Conversely, in vitro methylation of this site significantly reduced TPA induction. In vitro gel shift analysis revealed distinct and TPA-dependent binding of USF1 and USF2 to the repressor element that required nucleotides within the E-box. Furthermore, chromatin immunoprecipitation assay showed that USF, c-Myc, and Max proteins were associated with the FGF-BP promoter in vivo. Overall, these findings suggested that the balance between trans-activation by AP-1 and repression through the E-box is an important control mechanism for fine-tuning the angiogenic response to growth factor-activated pathways.

Induction of the angiogenic modulator fibroblast growth factor-binding protein by epidermal growth factor is mediated through both MEK/ERK and p38 signal transduction pathways.

Fibroblast growth factor-binding protein (FGF-BP) is a secreted protein that binds and activates fibroblast growth factors (FGF-1 and FGF-2) and induces angiogenesis in some human cancers. FGF-BP is expressed at high levels in squamous cell carcinoma (SCC) cell lines and tumor samples and has been shown to be rate-limiting for the growth of SCC tumors in vivo. In this study, we examine the regulation of FGF-BP by epidermal growth factor (EGF) and the signal transduction mechanisms that mediate this effect. We found that EGF treatment of the ME-180 SCC cell line caused a rapid induction of FGF-BP gene expression. This induction was mediated transcriptionally through the AP-1 (c-Fos/JunD) and CCAAT/enhancer-binding protein elements as well as through an E-box repressor site in the proximal regulatory region of the FGF-BP promoter. Pharmacological inhibition of protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) completely blocked EGF induction of FGF-BP mRNA, whereas inhibition of phosphatidylinositol 3-kinase had no effect. Additionally, both EGF- and anisomycin-induced FGF-BP mRNA was abrogated by inhibition of p38 mitogen-activated protein kinase, demonstrating a role for p38 in the regulation of FGF-BP. Co-transfection of the FGF-BP promoter with dominant negative forms of MEK2, extracellular signal-regulated kinase 2, and p38 significantly decreased the level of EGF induction, whereas expression of a dominant negative c-Jun N-terminal kinase mutant or expression of c-Jun N-terminal kinase inhibitory protein had no effect. Similarly, activation of the p38 pathway by overexpression of wild-type p38 or MKK6 enhanced FGF-BP transcription. These results demonstrate that EGF induction of FGF-BP occurs selectively through dual activation of the stress-activated p38 and the MEK/extracellular signal-regulated kinase mitogen-activated protein kinase pathways, which ultimately leads to activation of the promoter through AP-1 and CCAAT/enhancer-binding protein sites.

The expression of a novel, epithelium-specific ets transcription factor is restricted to the most differentiated layers in the epidermis.

Ets proteins have been implicated in the regulation of gene expression during a variety of biological processes, including growth control, differentiation, development and transformation. More than 35 related proteins containing the 'ets domain' have now been found which specifically interact with DNA sequences encompassing the core tetranucleotide GGAA. Although ets responsive genes have been identified in the epidermis, little is known about their distribution and function in this tissue. We have now demonstrated that epidermis and cultured epidermal keratinocytes synthesize numerous ets proteins. The expression of some of these proteins is regulated as a function of differentiation. Among these is a novel ets transcription factor with a dual DNA-binding specificity, which we have called jen. The expression of jen is not only epithelial specific, but it is the only ets protein so far described, and one of the very few transcription factors whose expression is restricted to the most differentiated epidermal layers. We show that two epidermal marker genes whose expression coincides with that of jen are transregulated by this protein in a complex mode which involves interactions with other transcriptional regulators such as Sp1 and AP1.