Three HPV-negative cell linesC33A, 293T, and HepG2 (Fig.5A)did not express 4-integrin. HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the 4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 VER-50589 effect. In summary, we identified a novel mechanism of human 4-integrin VER-50589 regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle. Human papillomaviruses (HPVs) are DNA viruses, which infect stratified epithelia of skin and mucosa. Depending on the virus type, they may give rise to a large spectrum of lesions ranging from benign skin warts to invasive carcinoma. Anogenital malignancies including cervical cancer and a group of head-and-neck squamous cell carcinomas are most commonly associated with high-risk mucosal HPV16 and HPV18 VER-50589 infection (38). For skin, the carcinogenic potential of HPV5 and HPV8 is fully accepted in epidermodysplasia verruciformis patients (24). Accumulating evidence supports a role of these viruses in the development of nonmelanoma skin cancer in the general population (36). Actively dividing basal cells of stratified epithelia are the VER-50589 target for viral infection but the productive viral life cycle takes place in suprabasal, differentiating cells (27). HPV replication critically depends on the coexistence of the cellular DNA replication machinery and differentiation events in keratinocytes (30). However, in normal keratinocytes these processes are separated and confined to either basal cells or the suprabasal differentiating cell compartment. While the viral E6 and E7 oncoproteins force infected cells to retain cell cycle capabilities and repress differentiation, it is unclear which signals commit infected keratinocytes to enter the differentiation process. The E2 protein is indispensable for both viral replication and gene expression. It acts predominantly as a DNA binding protein recognizing Rabbit Polyclonal to Cytochrome P450 26A1 the consensus palindromic sequence ACCN6GGT (27,30). Various studies have demonstrated that the papillomavirus E2 protein can display antiproliferative and prodifferentiation properties (4,15,19,21,33). Introduction of E2 from genital high-risk HPV into HPV-positive cancer cell lines results in repression of E6 and E7 oncogene transcription, leading to replicative VER-50589 senescence or apoptosis (13,19). Repression is the consequence of E2 binding to low-affinity E2 binding sites in the viral genome and displacement of cellular transcription factors from their binding sites, which are adjacent or overlap the E2 cognate sequences. This mode of transcriptional regulation seems to be a conserved property of E2, since it was shown for E2 proteins from high-risk mucosal (HPV16 and 18) and cutaneous (HPV8) virus types (5,12,14,43,46,48). Apart from regulatory effects on viral gene expression we also observed that HPV8 E2 repressed a cellular gene, 4-integrin, and this may be an early event to commit basal keratinocytes to enter suprabasal differentiation steps. Our data showed that E2 directly interacts with the gene regulatory region at three novel binding sites of the 4-integrin promoter. Detailed analysis defined that HPV8 E2 was able to displace a yet-unrecognized cellular factor from the second binding site (33). 4-Integrin is one of the major cell adhesion receptors in basal keratinocytes with fundamental functions in epithelial homeostasis. Expressed at the basolateral surface of keratinocytes, 4-integrin forms heterodimers with the 6 integrin subunit, and the 64 integrin binds to laminin-5. These specialized structures are crucial components of hemidesmosomes, which tightly attach basal cells to the underlying basement membrane and maintain proliferative potential (7). Since 4-integrin expression is undetectable in suprabasal keratinocytes, loss of 4-integrin expression may be among the first steps in keratinocyte differentiation, when cells leave the basal and enter the suprabasal layers (18). The molecular mechanism of how HPV8 E2 exactly represses 4-integrin expression remains poorly understood. We therefore aimed to identify the cellular factor displaced by E2 (E2-displaceable factor [E2-DF]) from the 4-integrin promoter. We show.
