The Human Papillomavirus Type 8 E6 Protein Interferes with NOTCH Activation during Keratinocyte Differentiation

Abstract

Cutaneous ␤-human papillomavirus (␤-HPV) E6 proteins inhibit NOTCH signaling by associating with the transcriptional coactivator MAML1. NOTCH has tumor suppressor activities in epithelial cells and is activated during keratinocyte differentiation. Here we report that HPV type 8 (HPV8) E6 subverts NOTCH activation during keratinocyte differentiation by inhibiting RBPJ/MAML1 transcriptional activator complexes at NOTCH target DNA. NOTCH inhibition impairs epithelial differentiation and may thus contribute to ␤-HPV replication and viral oncogenesis. Papillomaviridae are a large family of epitheliotropic viruses with double-stranded circular ϳ8-kb DNA genomes. More than 150 human papillomaviruses (HPVs) have been isolated (1). Some HPVs infect mucosal epithelia, whereas other HPVs infect cutaneous epithelial cells. HPV infections can be nonsymptomatic or cause hyperplastic skin lesions, commonly referred to as warts. Mucosal ␣-HPVs have been studied in great detail since some of these viruses, the "high-risk" HPVs, are the causative agents of cervical carcinoma as well as other anogenital cancers and oropharyngeal carcinomas. Cutaneous ␤-HPVs are less studied. While ␤-HPVs appear to be part of the normal "skin flora" and infections are often asymptomatic, they can cause cutaneous warts, which undergo malignant progression in patients suffering from the rare genetic disease epidermodysplasia verruciformis (EV) or in systemically immune-suppressed organ transplant patients, particularly in sun-exposed areas of the body (2-5). ␤-HPVs may also contribute to nonmelanoma skin cancers (NMSCs) in patients with a normal immune system, but this has not been formally proven.Cutaneous ␤-HPV E6 proteins have intrinsic transforming activities (6, 7) but do not associate with known cellular target proteins of high-risk mucosal ␣-HPV E6 proteins, including p53, UBE3A, and cellular PDZ proteins (8). Several groups have recently reported that the E6 proteins from HPV5, HPV8, and other ␤-HPVs can bind the NOTCH coactivator mastermind-like 1 (MAML1), thereby inhibiting NOTCH transcriptional programs (9-11). This is particularly interesting since NOTCH functions as a tumor suppressor in epithelial cells and mice with expression of a dominant negative MAML in basal epithelial cells develop squamous cell carcinoma (12). Since NOTCH signaling is important for cellular differentiation, we wanted to determine whether HPV8 E6 inhibits NOTCH signaling during keratinocyte differentiation. To experimentally address this issue, we generated populations of human keratinocytes immortalized with human telomerase reverse transcriptase (hTERT) (cl398) (13) and stably expressing amino-terminally hemagglutinin (HA)/FLAG epitopetagged HPV8 E6 (8E6-iHFKs) by lentiviral transduction. Vector-infected cells were generated as controls (C-iHFKs). To confirm the interaction of HPV8 E6 with components of the NOTCH transcriptional complex in these cells, we prepared lysates of 8E6-iHFKs and C-iHFKs in mammalian cell lysis buffer (MCLB; 50 mM Tris [pH 7.5], 150 mM...

NFX1-123 and Human Papillomavirus 16E6 Increase Notch Expression in Keratinocytes

“…These novel findings indicate consistent activation of the Notch signaling pathway, with increases in downstream targets driven by increased NFX1-123 that was reduced in parallel when NFX1-123 and Notch1 were decreased. Interestingly, the Notch1 transcription protein partner MAML is bound and targeted for degradation by beta-HPV E6 types to inhibit Notch signaling (50)(51)(52)(53); however, alpha-HPV E6 scramble short hairpin RNA control (scr), sh1RNA to NFX1-123 (sh1), or sh2RNA to NFX1-123 (sh2). Relative levels of Hes1 mRNA were calculated using the ⌬⌬C T method, normalizing mRNA levels to GAPDH within each sample.…”

Papillomaviruses

“…In addition, some beta-HPV E6 proteins have also been reported to cause degradation of the transcriptional coactivator p300, thereby short-circuiting DNA repair in response to UV damage by the Ataxia telangiectasia and Rad3-related kinase (Wallace et al, 2012;Howie et al, 2011). More recently, some beta-HPV E6 proteins have also been shown to inhibit NOTCH signaling, which is important for epithelial cell differentiation and stem cell maintenance (Meyers et al, 2013;Tan et al, 2012;Brimer et al, 2012).…”

The Viral Etiology of Skin Cancer

“…One of the critical regulators of epithelial differentiation is NOTCH signaling. Several recent studies have shown that the HPV5 and HPV8 E6 proteins inhibit NOTCH signaling by interacting with MAML proteins, critical co-activators of the NOTCH transcription complex (Brimer et al, 2012;Rozenblatt-Rosen et al, 2012;Tan et al, 2012;Meyers et al, 2013). NOTCH has tumor suppressor activities in epithelia, and inactivating NOTCH pathway mutations are highly prevalent in SCCs (Agrawal et al, 2011;Stransky et al, 2011).…”