Figure 1.
Langerin-positive cells and CCL20 expression are reduced in lesional skin of EV patients.
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.
Figure 2.
C/EBPβ binds to the enhancer region of CCL20 in vivo.
(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.
Figure 3.
Differentiation-specific nuclear C/EBPβ expression in human epidermis and C/EBPβ-mediated induction of CCL20 expression in NHK.
(A) Sections of normal human skin were stained using a polyclonal antibody against C/EBPβ (brown) and hematoxylin. A higher magnification of the complete epidermis is presented. Bars correspond to 100 µm and to 50 µm in the higher magnification. (B) NHK were co-transfected with the CCL20 promoter reporter construct and increasing amounts of C/EBPβ expression vector as indicated. Total amount of DNA was adjusted with the pcDNA3.1+ control vector. After 24 h the luciferase activity was measured and normalized to protein concentrations of the respective luciferase extracts. The normalized luciferase activity of the control transfection was set at 1. Shown are the values averaged from two transfections performed in triplicates ± SD. (C, D) RTS3b cells were transfected with C/EBPβ expression vector. After 24 h mRNA was isolated from the cells and quantified by real-time PCR for CCL20 in relation to GAPDH (C). Supernatants collected from these cell cultures were assessed for CCL20 protein expression by ELISA (D). Shown are the mean values ± SD from at least three independent experiments. Asterisks represent statistical significances, p<0.001. (E) Two promoter-proximal C/EBP binding sites are crucial for CCL20 activation. NHK were transfected with luciferase reporter constructs either under the control of the wild-type CCL20 promoter (black bars) or the CCL20 promoter mutated in one (white bars) or two (grey bars) proximal C/EBP binding sites and co-transfected with C/EBPβ (400 ng) expression vector. Total amount of DNA was adjusted with the pcDNA3.1+ control vector. After 24 h the luciferase activity was measured and normalized to protein concentration of the respective luciferase extracts. The normalized luciferase activity of the control transfection was set at 1. Shown are the values averaged from three transfections performed in triplicates ± SD. Asterisks represent statistical significances, p<0.0001.
Figure 4.
Endogenous C/EBPβ contributes to CCL20 expression in NHK.
(A) Nuclear extracts isolated from NHK stimulated with 50 ng/ml PMA for 6 h were analyzed for C/EBPβ expression by Western blot. Anti-HMGB1 antibodies were used as a control. Shown is one representative experiment out of three. (B, C) NHK were transfected with luciferase reporter constructs either under the control of the wild-type CCL20 promoter (black bars), the CCL20 promoter containing mutations of the proximal NF-κB binding site (white bars) or mutations in the promoter-proximal C/EBP binding sites (grey bars). 24 h post-transfection the cells were stimulated with 50 ng/ml PMA or medium as a control. 24 h later expression of the CCL20 protein was measured by ELISA in supernatants of NHK transfected with the wild-type CCL20 promoter construct (B), while in all transfected cells luciferase activity was determined (C). (D) NHK were co-transfected with the wild-type CCL20 promoter and increasing amounts of the LIP expression vector (0.6, 1.2 µg). Total amount of DNA was adjusted with pcDNA3.1+ vector. LIP expression was investigated in Western blot analysis, HMGB1 was used as loading control. In all reporter gene assays luciferase activity was measured 24 h after transfection and normalized to protein concentrations of the respective luciferase extracts. The normalized luciferase activity of the control transfection was set at 1. Shown are the values averaged from two transfections performed in triplicates ± SD. Asterisks represent statistical significances, p<0.0001.
Figure 5.
HPV8 E7 directly interacts with C/EBPβ and suppresses C/EBPβ-induced activation of the CCL20 promoter.
(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 (*).
Figure 6.
HPV8 E7 interferes with binding of C/EBPβ to the CCL20 promoter.
(A) Nuclear extracts from HaCaT cells stably expressing HPV8 E7 (pLXSN-HPV8 E7) and corresponding control cells (pLXSN) were analyzed by Western blot for C/EBPβ protein and HMGB1 expression (upper panels). Identical amounts of the respective nuclear extracts were used for EMSA using the 32P-labeled oligonucleotides (nt 734–748) containing the C/EBP binding site in the CCL20 promoter (lower panel). The complex corresponding to endogenous C/EBP binding activity within the CCL20 promoter is indicated by an arrow. (B) The same cells were used for chromatin immunoprecipitation. Protein-genomic DNA complexes were precipitated with anti-C/EBPβ antibody. DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter. The amplicon was quantified (lower panel) and visualized on an agarose gel (upper panel). The amount of target DNA precipitated from the pLXSN control cells was set at 100%. The mean values ± SD from three independent experiments are presented. Asterisks represent statistical significance, p = 0.008.
Figure 7.
HPV8 E7 suppresses CCL20 expression and Langerhans cell migration.
CCL20 mRNA (A) and protein (B) levels were quantified in NHK stably expressing HPV8 E6 (pLXSN-HPV8 E6) or E7 (pLXSN-HPV8 E7) oncogenes. The amount of CCL20 mRNA (in relation to β-actin as measured by quantitative real-time PCR) or protein in control cells was set at 1. CCL20 protein levels in supernatants collected from E6 or E7 expressing keratinocytes and corresponding control cells (pLXSN) were determined by ELISA. Measurements represent the mean values from three independent retroviral infections ± SD. Asterisks represent statistical significance, p<0.001. (C) Migration of langerin- and CCR6-positive (left panel) Langerhans cells was assessed in response to conditioned media collected from pLXSN control NHK in the presence or absence of anti-CCL20 antibody (Ab) or respective isotype control Ab and from NHK stably expressing HPV8 E6 (pLXSN-HPV8 E6) or E7 (pLXSN-HPV8 E7) oncogenes (right panel). Recombinant human CCL20 (rhCCL20) was used as a positive control. After 24 h transmigrated cells were counted. Data were collected in triplicates from at least two independent experiments using conditioned media from two independent retroviral infections. Shown are the mean values ± SD. Asterisks represent statistical significances, p<0.0001.
Figure 8.
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.