C/EBPβ binds to the enhancer region of CCL20 in vivo.

<p>(A) Nucleotide sequence of the human CCL20 promoter region with twelve putative C/EBP binding sites (underlined). Numbers below the underlined C/EBP binding sites mark the sequences, which display C/EBP DNA binding activity in EMSA. In bold is the DNA sequence tested for C/EBP binding in ChIP assay. (B) ³²P-labeled oligonucleotides containing the respective C/EBP binding sites (nt 294–308, nt 574–584, nt 652–667, nt 716–724, nt 734–748) of the CCL20 promoter were incubated with 5 µg GST, GST-C/EBPα or GST-C/EBPβ fusion proteins and analyzed by EMSA. The arrow indicates complexes corresponding to C/EBP DNA binding activity. (C) Chromatin immunoprecipitation assay was performed using RTS3b cells transfected with the C/EBPβ expression vector. For precipitation anti-C/EBPβ (H-7) antibody was used. Genomic DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter (in bold). The amplicon was quantified (left panel) and visualized on an agarose gel (right panel). The amount of target DNA precipitated with the control antibody was set at 1. Shown are mean values ± SD from four experiments. The asterisk represents statistical significance, p = 0.02.</p

Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

<div><p>Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of <em>CCL20</em> gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive <em>CCL20</em> gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.</p> </div

Langerin-positive cells and CCL20 expression are reduced in lesional skin of EV patients.

<p>Sections of normal human skin (A, B, C) and lesional skin from EV patients (D–J) were stained using antibodies against langerin (A, D) or CCL20 (B, C, E, F) and hematoxylin. To verify the specificity of the immunohistochemical reaction, recombinant CCL20 protein was used in a blocking experiment (C). Lesion positive for HPV8 only (D, E, G–J). Lesion coinfected with HPV5 and HPV8 (F). Validation of active infection was visualized by hematoxylin eosin staining to reveal the characteristic E4 cytoplasmic inclusions typically found in EV lesions (G) along with immunofluorescence staining for the HPV8 E4 protein (green (H)) and FISH staining to identify the presence of amplified HPV genomes (red (I)). The merged image showing the onset of genome amplification in E4-positive cells is shown in J. Cell nuclei are visualized in J by counterstaining with DAPI. Selected cells double positive for E4 protein and HPV8 DNA are marked with arrows. Bars correspond to 100 µm in A–E and G–J, and to 200 µm in F.</p

Schematic presentation of the molecular mechanism how human papillomavirus type 8 interferes with a novel C/EBPβ-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.

<p>Schematic presentation of the molecular mechanism how human papillomavirus type 8 interferes with a novel C/EBPβ-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.</p

HPV8 E7 directly interacts with C/EBPβ and suppresses C/EBPβ-induced activation of the CCL20 promoter.

<p>(A) NHK were transfected with CCL20 promoter luciferase construct (0.5 µg) and C/EBPβ (0.4 µg) in the presence or absence of HPV8 E6 (0.8 µg) or HPV8 E7 (0.8 µg) pcDNA3.1+ expression vectors. Total amount of DNA was adjusted with empty vector. After 24 h the luciferase activity was measured and normalized to protein concentration of the respective luciferase extract. The normalized luciferase activity of the control transfection was set at 1. Transfections were conducted in triplicates. Shown are mean values from three independent experiments ± SD. Asterisks represent statistical significance, p<0.001. (B) HPV8 E7 co-localizes with C/EBPβ in the keratinocyte nucleus. RTS3b cells seeded on glass coverslips were co-transfected with Flag-HPV8 E7 and ECFP-C/EBPβ expression vectors and stained with DAPI (blue, first panel) as well as anti-Flag antibody (red, second panel). ECFP-C/EBPβ is shown in third panel (green). Cells were analyzed by deconvolution fluorescence microscopy. The overlays and co-localization (yellow) are displayed in the fourth panel. Bars correspond to 20 µm. (C) In pull-down assays GST, GST-p300, GST-C/EBPα or GST-C/EBPβ were incubated with in vitro-translated HPV8 E7 protein (IVT, input) and precipitated (P) by glutathione Sepharose beads. pcDNA3.1+ vector (control vector) was used as a control for HPV8 E7 in vitro translation. Proteins were visualized by SDS-PAGE and autoradiography. (D) C33A cells were transfected with pFlag-CMV2, the Flag-tagged HPV8 E7 construct or deletion mutants thereof and co-transfected with the C/EBPβ expression vector. After 24 h cell lysates were prepared and precipitated with anti-Flag agarose beads (IP). The precipitates (P) and input (I, 10 µl of the lysates) were analyzed with anti-C/EBPβ or anti-Flag antibodies by Western blot (WB). The anti-Flag antibody light chain present in the precipitates is marked with a star (*).</p

Data_Sheet_3.DOCX

<p>Persistent genus β-HPV (human papillomavirus) infection is a major co-factor for non-melanoma skin cancer in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). Malignant EV lesions are particularly associated with HPV type 5 or 8. There is clinical and molecular evidence that HPV8 actively suppresses epithelial immunosurveillance by interfering with the recruitment of Langerhans cells, which may favor viral persistence. Mechanisms how persistent HPV8 infection promotes the carcinogenic process are, however, less well understood. In various tumor types chronic inflammation has a central role in tumor progression. The calprotectin complex consisting of S100A8 and S100A9 proteins has recently been identified as key driver of chronic and tumor promoting inflammation in skin carcinogenesis. It induces chemotaxis of neutrophil granulocytes and modulates inflammatory as well as immune responses. In this study, we demonstrate that skin lesions of EV-patients are massively infiltrated by inflammatory cells, including CD15⁺ granulocytes. At the same time we observed a very strong expression of S100A8 and S100A9 proteins in lesional keratinocytes, which was mostly confined to the suprabasal layers of the epidermis. Both proteins were hardly detected in non-lesional skin. Further experiments revealed that the HPV8 oncoproteins E6 and E7 were not involved in S100A8/A9 up-regulation. They rather suppressed differentiation-induced S100A8/A9 expression. In contrast, the viral transcription factor E2 strongly enhanced PMA-mediated S100A8/A9 up-regulation in primary human keratinocytes. Similarly, a tremendous up-regulation of both S100 proteins was observed, when minute amounts of the PMA-inducible CCAAT/enhancer binding protein β (C/EBPβ), which is expressed at low levels in the suprabasal layers of the epidermis, were co-expressed together with HPV8 E2. This confirmed our previous observation that C/EBPβ interacts and functionally synergizes with the HPV8 E2 protein in differentiation-dependent gene expression. Potent synergistic up-regulation of S100A8/A9 was seen at transcriptional and protein levels. S100A8/A9 containing supernatants from keratinocytes co-expressing HPV8 E2 and C/EBPβ significantly induced chemotaxis of granulocytes in migration assays supporting the relevance of our finding. In conclusion, our data suggest that the HPV8 E2 protein actively contributes to the recruitment of myeloid cells into EV skin lesions, which may support chronic inflammation and progression to skin cancer.</p

Data_Sheet_1.DOCX

<p>Persistent genus β-HPV (human papillomavirus) infection is a major co-factor for non-melanoma skin cancer in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). Malignant EV lesions are particularly associated with HPV type 5 or 8. There is clinical and molecular evidence that HPV8 actively suppresses epithelial immunosurveillance by interfering with the recruitment of Langerhans cells, which may favor viral persistence. Mechanisms how persistent HPV8 infection promotes the carcinogenic process are, however, less well understood. In various tumor types chronic inflammation has a central role in tumor progression. The calprotectin complex consisting of S100A8 and S100A9 proteins has recently been identified as key driver of chronic and tumor promoting inflammation in skin carcinogenesis. It induces chemotaxis of neutrophil granulocytes and modulates inflammatory as well as immune responses. In this study, we demonstrate that skin lesions of EV-patients are massively infiltrated by inflammatory cells, including CD15⁺ granulocytes. At the same time we observed a very strong expression of S100A8 and S100A9 proteins in lesional keratinocytes, which was mostly confined to the suprabasal layers of the epidermis. Both proteins were hardly detected in non-lesional skin. Further experiments revealed that the HPV8 oncoproteins E6 and E7 were not involved in S100A8/A9 up-regulation. They rather suppressed differentiation-induced S100A8/A9 expression. In contrast, the viral transcription factor E2 strongly enhanced PMA-mediated S100A8/A9 up-regulation in primary human keratinocytes. Similarly, a tremendous up-regulation of both S100 proteins was observed, when minute amounts of the PMA-inducible CCAAT/enhancer binding protein β (C/EBPβ), which is expressed at low levels in the suprabasal layers of the epidermis, were co-expressed together with HPV8 E2. This confirmed our previous observation that C/EBPβ interacts and functionally synergizes with the HPV8 E2 protein in differentiation-dependent gene expression. Potent synergistic up-regulation of S100A8/A9 was seen at transcriptional and protein levels. S100A8/A9 containing supernatants from keratinocytes co-expressing HPV8 E2 and C/EBPβ significantly induced chemotaxis of granulocytes in migration assays supporting the relevance of our finding. In conclusion, our data suggest that the HPV8 E2 protein actively contributes to the recruitment of myeloid cells into EV skin lesions, which may support chronic inflammation and progression to skin cancer.</p

Data_Sheet_4.DOCX

<p>Persistent genus β-HPV (human papillomavirus) infection is a major co-factor for non-melanoma skin cancer in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). Malignant EV lesions are particularly associated with HPV type 5 or 8. There is clinical and molecular evidence that HPV8 actively suppresses epithelial immunosurveillance by interfering with the recruitment of Langerhans cells, which may favor viral persistence. Mechanisms how persistent HPV8 infection promotes the carcinogenic process are, however, less well understood. In various tumor types chronic inflammation has a central role in tumor progression. The calprotectin complex consisting of S100A8 and S100A9 proteins has recently been identified as key driver of chronic and tumor promoting inflammation in skin carcinogenesis. It induces chemotaxis of neutrophil granulocytes and modulates inflammatory as well as immune responses. In this study, we demonstrate that skin lesions of EV-patients are massively infiltrated by inflammatory cells, including CD15⁺ granulocytes. At the same time we observed a very strong expression of S100A8 and S100A9 proteins in lesional keratinocytes, which was mostly confined to the suprabasal layers of the epidermis. Both proteins were hardly detected in non-lesional skin. Further experiments revealed that the HPV8 oncoproteins E6 and E7 were not involved in S100A8/A9 up-regulation. They rather suppressed differentiation-induced S100A8/A9 expression. In contrast, the viral transcription factor E2 strongly enhanced PMA-mediated S100A8/A9 up-regulation in primary human keratinocytes. Similarly, a tremendous up-regulation of both S100 proteins was observed, when minute amounts of the PMA-inducible CCAAT/enhancer binding protein β (C/EBPβ), which is expressed at low levels in the suprabasal layers of the epidermis, were co-expressed together with HPV8 E2. This confirmed our previous observation that C/EBPβ interacts and functionally synergizes with the HPV8 E2 protein in differentiation-dependent gene expression. Potent synergistic up-regulation of S100A8/A9 was seen at transcriptional and protein levels. S100A8/A9 containing supernatants from keratinocytes co-expressing HPV8 E2 and C/EBPβ significantly induced chemotaxis of granulocytes in migration assays supporting the relevance of our finding. In conclusion, our data suggest that the HPV8 E2 protein actively contributes to the recruitment of myeloid cells into EV skin lesions, which may support chronic inflammation and progression to skin cancer.</p

HPV8 E6 involves the miR203/p63-pathway to reprogram keratinocyte functions.

<p><b>(A)</b> Cell cycle analysis of HPV8 E6 expressing and pLXSN control HaCaT cells by flow cytometry using propidium iodide staining. Shown are cell counts in the respective cell cycle phases in relation to the gated single cell population. <b>(B)</b> BrdU incorporation was measured in triplicates in HPV8 E6 expressing HaCaT cells and normalized to pLXSN-control. <b>(C)</b> HPV8 E6 expressing and pLXSN control HaCaT monolayer cultures were scratched. Pictures were taken at time points 0, 24 and 48 h. The area of the gap, indicated by dotted lines (left panel), was determined in relation to time point 0 h (right panel). HPV8 E6 expressing cells were scratched 24 h after 10 nM p63-siRNA (or si-control) <b>(D),</b> or 20 nM miR-203-mimic transfection (or control-mimic) <b>(E)</b> and analyzed as in (B). Scale bar: 200 μm. Mean values ± SD from <i>n</i> = 3 experiments are shown. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)</p

p63 and miR-203 expression are altered in HPV8 E6 expressing HaCaT cells.

<p>HaCaT cells stably expressing HPV8 E6 or corresponding pLXSN control cells were investigated in different experiments. <b>(A)</b> p63 protein expression was analyzed by Western blot in relation to actin (shown is one experiment out of three), <b>(B)</b> ΔNp63α mRNA expression by qRT-PCR in relation to RPL13A. <b>(C)</b> Organotypic cultures were stained for p63 expression (brown). <b>(D)</b> Three independent cultures were quantified for p63-positive and -negative nuclei (per microscopic field in randomly selected areas, 200x). <b>(E)</b> MiR-203 expression was determined after treatment with Ca²⁺ for 72 h by Northern blot. 28S RNA served as loading control. One experiment out of three is shown. <b>(F)</b> The same RNA as in (E) was used to analyze miR-203 levels by qRT-PCR. Expression levels were normalized on RNU6B by 2^ΔΔCt. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (**p<0.01, ***p<0.001, ****p<0.0001)</p