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

p300 regulates C/EBPα and miR-203 expression.

<p><b>(A)</b> NHK stably expressing HPV8 E6, mutant Δ8E6 (Δaa132-136) deficient in p300-binding, or pLXSN control cells were analyzed for C/EBPα expression by qRT-PCR. <b>(B)</b> C/EBPα protein expression was determined by IHC in HPV8 E6, Δ8E6 expressing HaCaT or control organotypic cultures (red). <b>(C)</b> In retrovirally infected NHK cells miR-203 expression was measured by qRT-PCR, <b>(D)</b> p63 and involucrin protein expression was determined by Western blot in relation to actin (shown is one representative blot out of three). <b>(E)</b> ΔNp63α and <b>(F)</b> involucrin mRNA expression levels were measured by qRT-PCR normalized to RPL13A. <b>(G)</b> NHK were transfected with 10 nM p300-specific siRNA (pool or two single siRNAs versus si-control), harvested 48 h later and expression of p300 mRNA, miR-203, C/EBPα and C/EBPβ mRNA were quantified by qRT-PCR as described. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicate are shown. <b>(H)</b> HPV8 E6 expressing HaCaT or pLXSN control cells were subjected to chromatin immunoprecipitation. Protein-genomic DNA complexes were precipitated with anti-p300 antibody. DNA was isolated, amplified by real-time PCR with primers specific for the nt -1533 to -1398 regulatory region of the <i>CEBPΑ</i> gene. The amount of target DNA precipitated with the isotype control antibody was set at 1. The mean values ± SD from 3 independent experiments are presented. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</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 regulation in EV-lesions and by HPV8 oncoproteins in NHK in vitro.

<p>Sections of non-lesional skin <b>(A, C)</b> and HPV8-positive lesional skin <b>(B, D)</b> from EV-patients were stained using antibodies against p63 (A, B) or probes against miR-203 (C, D). NHK stably expressing HPV8 E6, E7, E6/E7 or the corresponding pLXSN control cells were analyzed for <b>(E)</b> p63 protein expression by Western blot in relation to actin expression (shown is one experiment out of three), <b>(F)</b> ΔNp63α mRNA or <b>(G)</b> miR-203 expression by qRT-PCR. ΔNp63α mRNA was determined in relation to RPL13A, miR-203 level to RNU6B by the 2^ΔΔCt method. In (F, G) mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (**p<0.01, ***p<0.001, ****p<0.0001)</p

C/EBPα is a direct transcriptional regulator of miR-203.

<p><b>(A-C)</b> NHK stably expressing HPV8 E6 or corresponding pLXSN control cells were stimulated with 50 ng/ml PMA for 24 h. <b>(A)</b> miR-203 levels were measured in qRT-PCR and normalized to RNU6B by the 2^ΔΔCt method. <b>(B)</b> ΔNp63α and <b>(C)</b> involucrin mRNA expression levels were measured by qRT-PCR and normalized to RPL13A. NHK pLXSN control cells were analyzed for C/EBPα mRNA expression <b>(D)</b> 24 h after 50 ng/ml PMA or <b>(E)</b> 72 h after 1.2 mM Ca²⁺ stimulation. <b>(F)</b> NHK were transfected with 10 nM siRNA (pool or two single siRNAs) against C/EBPα (or si-control). C/EBPα mRNA or miR-203 expression levels were determined by qRT-PCR. <b>(G)</b> ³²P-labeled oligonucleotides containing a C/EBP binding site from the IL-6 gene (control) or a predicted binding site within the miR-203 hairpin sequence (miR-203 BS, nt +57 to +63) or its mutated form (MUT) were incubated with 5 μg (control) or 20 μg (miR-203 BS, MUT) nuclear extracts (NE) from C33a cells transfected with pcDNA3.1-C/EBPα expression vector and analyzed by EMSA. For supershift analysis, nuclear extracts were pre-incubated with 2 μg of two different antibodies against C/EBPα (clone 14AA and N19) or respective isotype control. Asterisk indicates supershifted bands. Gel slots are indicated by an arrow. <b>(H)</b> NHK were transfected with 10 nM siRNA (pool or two single siRNAs) directed against C/EBPα (or si-control) 24 h before transfection of the miR-203 promoter reporter construct comprising the miR-203 regulatory region (-1165 nt to +70). Cells were stimulated with 50 ng/ml PMA for 24 h. Luciferase activity was measured and normalized to protein concentrations of the respective luciferase extracts. <b>(I, J)</b> HaCaT cells were co-transfected with 0 or 100 ng C/EBPα expression vector and wildtype (WT) miR-203 reporter (in I from -1165 nt to +70 nt; in J from +47 nt to +73 nt) or respective mutated miR-203 reporter constructs (MUT, comprising the mutated C/EBPα binding site within the miR-203 hairpin sequence) and luciferase activity was determined. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p

Differentiation suppression by HPV8 E6 involves the miR203/p63-pathway.

<p>Organotypic cultures generated from <b>(A)</b> HPV8 E6 expressing or pLXSN control HaCaT cells were stained for involucrin expression (brown). <b>(B,C)</b> HaCaT cells were stimulated with Ca²⁺ for 72 h. <b>(B)</b> Involucrin protein expression was determined by Western blot in relation to actin (shown is one experiment out of three), <b>(C)</b> involucrin mRNA expression by qRT-PCR in relation to RPL13A. <b>(D, E, F)</b> HPV8 E6 expressing and pLXSN control HaCaT cells were transfected with 10 nM p63 specific siRNA (or si-control). <b>(D)</b> 48 h post transfection, p63 and involucrin protein expression levels were determined by Western blot in relation to actin expression (shown is one experiment out of three), <b>(E)</b> ΔNp63α and <b>(F)</b> involucrin mRNA expression by qRT-PCR in relation to RPL13A. <b>(G, H, I)</b> HPV8 E6 expressing HaCaT cells were transfected with 20 nM hsa-miR-203-mimic (or control-mimic). 72 h post transfection cells were analyzed as in D-F. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)</p