Epidermal growth factor induction of human papillomavirus type 16 E6/E7 mRNA in tumour cells involves two AP-1 binding sites in the viral enhancer

Abstract

The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papiUomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell typespecific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragrnents with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.

Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells

“…When a 4 bp mutation in the AP-1 motif in the promoter was tested in PC12 cells, the results clearly demonstrated that this motif was required for the induction of the clusterin promoter by both NGF and EGF. Moreover, the mutant gave a weaker basal expression than the wild type, which confirms previous findings that AP-1 sites can contribute to basal promoter activity in the absence of inducers [39,40]. EMSA (' band shift ') studies to identify the protein(s) interacting with the sequence motif demonstrated binding of a factor to the AP-1 motif that was competed for by the homologous sequence and by two other AP-1 sites, including the site from the collagenase promoter, but not by other fragments not containing an AP-1 site.…”

“…AP-1 activation can also be achieved by the stimulation of protein kinase C with phorbol esters, which elicits an signalling response via the Raf-1\MEK\ERK cascade [24,44]. Most AP-1 binding sites have been functionally tested by phorbol ester stimulation of protein kinase C : only in very few cases have AP-1 sites been characterized as EGF response elements [24,40,45]. It can be assumed, however, that the stimulation of a whole set of genes by EGF, NGF and other growth factors goes through the activation of AP-1.…”

The EVER Proteins as a Natural Barrier against Papillomaviruses: a New Insight into the Pathogenesis of Human Papillomavirus Infections

“…Furthermore, there are multiple AP-1-binding sites within the LCR of the HPV genome ( Fig. 1) (111, 162, 203) that enhance E6/E7 expression (discussed below) (111,136,147,162). Moreover, AP-1 contributes significantly to HPVmediated carcinogenesis (119), and the composition of AP-1 clearly changes during the course of the transformation process (170,197).…”

Identification of a novel promoter in the E2 open reading frame of the human papillomavirus type 18 genome

“…Also, Meyers et al [1997] demonstrated these additional HPV-18 splice sites by studying HCK keratinocyte cell lines carrying episomal HPV-18: donor site at nucleotide 233 and acceptor site at nucleotide 2779 resulting in the deletion of E6, E7, and E1 ORFs; and donor site at nucleotide position 929 and acceptor site at nucleotide position 3434 resulting in the deletion of E1 and part of E2. An explanation proposed by Peto et al [1995] for a single band having to account for transcripts of different sizes is that bands migrating near the ribosomal RNA are not separated due to compression by the ribosomal RNA.…”