The human papillomavirus type 8 E6 protein interferes with NOTCH activation during keratinocyte differentiation - PubMed (original) (raw)

The human papillomavirus type 8 E6 protein interferes with NOTCH activation during keratinocyte differentiation

Jordan M Meyers et al. J Virol. 2013 Apr.

Abstract

Cutaneous β-human papillomavirus (β-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 β-HPV replication and viral oncogenesis.

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Figures

Fig 1

HPV8 E6 biochemically and functionally interacts with the NOTCH signaling pathway in immortalized human foreskin keratinocytes (iHFKs). (A) HPV8 E6 complexes with NOTCH transcription components in iHFK cells. Lysates from control and HPV8 E6-expressing iHFKs (C-iHFKs and 8E6-iHFKs, respectively) were collected after 6 days of calcium treatment. Input corresponds to 5% used for immunoprecipitations. HPV8 E6 immunoprecipitated with HA antibody associates with both MAML1 and intracellular NOTCH (ICN). α, anti. (B) RNA isolated from C-iHFKs and 8E6-iHFKs grown in keratinocyte serum-free medium (KSFM) was analyzed for expression of MAML1 and HES by quantitative reverse transcription-PCR (qRT-PCR). Expression of the NOTCH target gene HES1 was lower in 8E6-iHFKs than in C-iHFKs (0.60 ± 0.09; P = 0.0001) despite the fact that the NOTCH coactivator MAML1 was expressed at higher levels in 8E6-iHFKs than in C-iHFKs (1.38 ± 0.12; P = 0.0006). The graph depicts averages and standard deviations of 4 independent experiments, each performed in duplicate.

Fig 2

Calcium-induced differentiation of iHFKs activates NOTCH signaling, which is inhibited in HPV8 E6-expressing iHFKs. C-iHFKs (A) and 8E6-iHFKs (B) were grown to 80% confluence in KSFM and then switched to Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum. RNA was isolated at various times, and HES1 and MAML1 mRNA levels were measured by qRT-PCR. HES1 and MAML1 mRNA levels dramatically increase upon calcium treatment of C-iHFKs, whereas there is only a slight increase in HES1 and MAML1 mRNA expression in 8E6-iHFKs. A representative experiment is shown; similar results were obtained in 2 additional experiments. Error bars show standard deviations from technical replicates of the qRT-PCR experiment shown. (C) NOTCH inhibition by a gamma secretase inhibitor (2 μM CpdE-GSI) abrogates induction of HES1 mRNA expression and HPV8 E6 expression similarly at both day 0 and day 6 after calcium addition. Preparation of cells was similar to that for panels A and B except for CpdE and DMSO (vehicle) addition as shown. *, P < 0.05 compared to results for C-iHFK (DMSO). Data represent averages and standard deviations from two independent experiments.

Fig 3

Immunoblot analysis of NOTCH signaling components and bona fide NOTCH-regulated genes. Protein extracts from C-iHFKs and 8E6-iHFKs treated with either DMSO or 2 μM CpdE (GSI) were prepared at the indicated times from calcium-differentiated C-HFKs and 8E6-iHFKs. Steady-state levels of various NOTCH signaling-associated proteins were quantified using ImageJ software. Data shown are representative of two independent experiments.

Fig 4

Comparative analysis of NOTCH signaling pathway components in calcium-differentiated 8E6-iHFKs and GSI-treated C-HFKs by qRT-PCR. 8E6-iHFKs treated with DMSO and C-iHFKs treated with either DMSO or CpdE (2 μM) were grown for 6 days in calcium differentiation medium, and total RNA was collected and analyzed using the human NOTCH signaling pathway PCR array (SABiosciences). Genes corresponding to those identified in the KEGG NOTCH pathway are displayed, with relative mRNA levels compared to those for differentiated C-iHFKs treated with DMSO indicated using the color scheme shown at the bottom. Results represent averages from duplicate PCR arrays.

Fig 5

HPV8 E6 accesses regulatory regions of NOTCH target genes and is dependent in part on ICN. (A) Schematic of the different transcriptional states of NOTCH target genes (NTG). In the repressed state, NOTCH-responsive elements are occupied by RBPJ bound to corepressors (Co-R; left), and in the active state, ICN is bound to RBPJ, a MAML family member, and other coactivators (Co-A) such as p300/CBP (middle). Association of HPV8 E6 with MAML may prevent formation of the activator complex and thereby preserve the repressor complex, or HPV8 E6 may bind to the activator complex and interfere with its transcriptional activation activity (right). The results shown are consistent with the second model. (B) Schematic of RBPJ binding sites in the HES1 regulatory region. Primers amplify tandem RBPJ binding sites in a promoter-proximal region as well as a single RBPJ binding site upstream of the promoter. (C) C-iHFKs and 8E6-iHFKs were harvested and fixed with formaldehyde. Sonicated chromatin was immunoprecipitated with anti-HA antibody (Abcam), and after reversing cross-links purified DNA was analyzed by qPCR. The HA signal is represented as fold increase over control IgG antibody with promoter-proximal (P) and enhancer (E) primer sets. (D) ChIP was performed on 8E6-iHFKs treated with either DMSO or GSI (2 μM CpdE) and analyzed by qPCR. The HA signal decreases with GSI treatment, along with modest reductions in Pol II and RBPJ. Results shown are representative of two independent experiments.

References

1. Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen H, de Villiers EM. 2010. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 401:70–79 - PMC - PubMed
1. Dubina M, Goldenberg G. 2009. Viral-associated nonmelanoma skin cancers: a review. Am. J. Dermatopathol. 31:561–573 - PubMed
1. Lazarczyk M, Cassonnet P, Pons C, Jacob Y, Favre M. 2009. The EVER proteins as a natural barrier against papillomaviruses: a new insight into the pathogenesis of human papillomavirus infections. Microbiol. Mol. Biol. Rev. 73:348–370 - PMC - PubMed
1. Meyer T, Arndt R, Nindl I, Ulrich C, Christophers E, Stockfleth E. 2003. Association of human papillomavirus infections with cutaneous tumors in immunosuppressed patients. Transpl. Int. 16:146–153 - PubMed
1. Pfister H. 2003. Chapter 8: human papillomavirus and skin cancer. J. Natl. Cancer Inst. Monogr. 2003:52–56 - PubMed

The human papillomavirus type 8 E6 protein interferes with NOTCH activation during keratinocyte differentiation - PubMed (original) (raw)