%0 Generic
%A Mühlen, Sabrina
%D 2008
%F heidok:8815
%K Humanes Papillomvirus , Frühe Proteine , MAP-KinasenHuman papillomavirus , early proteins , MAP-kinases , signal trnasduction , oncology
%R 10.11588/heidok.00008815
%T Influence of Papillomavirus Early Proteins on the Expression of Tumor-Progression Promoting Genes
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8815/
%X Human papillomaviruses (HPV) are the known cause for cancers of the cervix and are associated with a variety of other human malignancies including head and neck, and skin. The cottontail-rabbit papillomavirus (CRPV) serves as a suitable animal model to study the development and progression of these cancers in-vivo. Our group has previously demonstrated that CRPV-induced skin lesions express elevated levels of metalloproteinase-9, a protease contributing to cancer progression by extracellular matrix remodelling. Based on our previous findings that the CRPV early protein 2 (E2) can activate a truncated human MMP-9 promoter fragment, we hypothesized that enhanced MMP-9 expression in the rabbit lesions is a consequence of activation of the rabbit MMP-9 promoter by CRPV E2. In order to elucidate the mechanism involved in MMP-9 promoter activation a library of genomic DNA isolated from rabbit skin was constructed, and the sequence of the MMP-9 promoter was identified. Promoter deletion mutants were cloned and the minimum required fragment for E2-mediated MMP-9 promoter activation was determined to be -717 bp in length. Selective mutation of transcription factor binding sites within the promoter sequence revealed a high importance of both of the two identified AP-1 binding sites in the rabbit MMP-9 promoter. Using the transactivation-deficient c-Jun mutant TAM67, a strong inhibitory effect on promoter activation after challenge with E2 could be observed, suggesting an important role of c-Jun in the activating AP-1 transcription-factor complex. The same mechanism could be shown in the human system during this study. Furthermore, it could be determined that in both, the rabbit and the human system, the activation of the MMP-9 promoter by E2 requires the phosphorylation of the MAP-kinase ERK, as inhibition of the cascade by the chemical inhibitor PD098059 resulted in a significant decrease of promoter activation. Co-transfection of E2 and siRNA directed against ERK or a dominant-negative mutant of the latter led to similar results.  It has been described previously that the high-risk HPV E2 is located within both nucleus and cytoplasm. Mutations in the domains responsible for protein localization allowed for investigation of the role of E2 localization in the activation of the MMP-9 promoter. It was observed that MMP-9 activation was strikingly decreased when the protein was mainly cytoplasmic. Additionally, HPV6bE2 which has been described to be solely nuclear did also induce MMP-9 promoter activation.  It can hence be concluded that CRPV E2 and HPV16 E2 both activate the respective MMP-9 promoters via an AP-1 and ERK dependent mechanism. As direct binding of the E2 proteins to the promoter can be roled out, this mechanism has to be further investigated. It can, however, be hypothezised that the interaction of E2 with its potential interation partner is taking place within the nucleus.