In this study, we found that the HPV 16 E6 and E7 oncoproteins caused a synergistic down-regulation of endogenous IVL mRNA and protein levels in HFK cells, which are natural host cells of the virus. Our finding is in accordance with previous studies performing microarray analysis of genes involved in cervical carcinogenesis. IVL and/or other keratinocyte differentiation associated genes (such as certain keratins and small prolin-rich proteins) are down-regulated in cervical cancer specimens compared to normal cervical samples [20, 21]. Accordingly, several studies using cultured human keratinocytes as in vitro models for cervical carcinogenesis found that the expression of HPV oncogenes causes a down-regulation of expression of IVL and/or other genes involved in epithelial differentiation [13, 22, 23]. However, microarray analysis does not provide information on the mechanism of changes in gene expression. Therefore, our approach was to analyse in HFK cells gene expression alterations of a few selected genes involved in keratinocyte differentiation using reliable real-time RT-PCR assays and to explore the molecular mechanisms behind gene expression alterations using luciferase reporter assays. We also found it important to study the effects of the HPV oncogenes on the expression of differentiation-regulated genes both in proliferating and in differentiating HFK cells as we thought that differentiating cells rather than proliferating cells reflect better the cellular environment required for the productive life cycle of HPV.
As expected, induction of differentiation of keratinocytes highly increased the endogenous mRNA levels of both IVL and TG1 in HFK cells (Figure 2). Interestingly, the HPV 16 E6 and E7 oncogenes together had a very strong down-regulating effect on IVL mRNA but only a moderate effect on TG1 mRNA. This suggests that the HPV oncogenes may have different effects on the expression of different genes involved in the differentiation of squamous epithelial cells. Western blot analysis showed that the joint effect of HPV 16 E6 and E7 on transcriptional down-regulation resulted in excessive decrease of IVL protein levels as well, both in proliferating and in differentiating cells (Figure 3). In a previous study, the expression of HPV 6 or HPV 16 E7 was shown to result in a decrease of IVL protein levels in HFK cells . We can conclude that the HPV 16 E6 and E7 oncogenes together seem to down-regulate basal IVL expression and also decrease the differentiation-induced expression of the IVL gene in HFK cells.
The expression of genes involved in keratinocyte differentiation (including IVL) are generally regulated on the level of transcription . Therefore, it seemed reasonable to investigate the effects of HPV 16 oncoproteins on IVL promoter activity. This approach included transfecting HFK cells by HPV 16 E6 and/or E7 expression vectors along with luciferase reporter constructs containing the whole upstream-regulatory region (URR) of the human IVL gene. In agreement with previous results , differentiation of HFK cells led to a significant increase in the transcriptional activity of the IVL promoter. In proliferating HFK cells, HPV 16 E6, but not E7 caused a significant down-regulation of IVL promoter activity. The HPV 16 E6 and E7 oncoproteins together caused a down-regulation of IVL promoter activity in differentiating HFK cells (Figure 4). Taken together, these results suggest that the down-regulation of endogenous IVL mRNA and protein levels in HFK cells by the HPV 16 E6 oncoprotein is caused by inhibition of IVL promoter activity. However, it can not be ruled out that HPV 16 E6 down-regulates the expression of IVL or other differentiation-associated genes also by other mechanisms. For example, HPV 16 E6 was shown to down-regulate the expression of Notch1, which was suggested to have a role in the suppression of keratinocyte differentiation by E6 .
In order to localise the effect of the HPV oncogenes within the IVL promoter, we made luciferase reporter constructs containing different parts of the URR of the human IVL gene. The URR of the human IVL gene contains a distal regulatory region (DRR, -2473/-1953 from transcription start site) and a proximal regulatory region (PRR, -241/-7 from the transcription start site) . From the 5 possible AP1 (activator protein 1) binding sites in the URR, AP1-5 (in DRR) and AP1-1 (in PRR) are essential for optimal promoter activity . AP1 factors (c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD) are expressed at specific epidermal layers and the expression pattern of these factors is thought to have a role in differentiation-regulated gene expression in keratinocytes [25, 29, 30]. Fra-1, junB and junD interact with AP1 sites within the human IVL promoter and mediate phorbol ester responsiveness . In our experiments, the level of inhibition by HPV 16 E6 was the highest for the construct containing the whole URR of the IVL gene, but an IVL reporter construct carrying only the PRR was still significantly inhibited by the HPV 16 E6 protein, both in proliferating and in differentiating HFK cells (Figure 5). This suggests that the PRR of IVL gene contains binding sites for transcription factors that are regulated by HPV 16 E6.
HPV 16 E7 had a significant inhibitory effect only on the construct containing the full-length IVL promoter (IVL 2418), and this effect was seen only in differentiating cells (Figure 5). This may suggest that the effect of E7 on the IVL promoter is less direct and/or less specific than that of E6. We find it conceivable that the effects of E7 seen on IVL expression (synergistic down-regulating effect with E6) and on IVL promoter (slight down-regulation only in differentiating cells) are caused not by a direct and specific interaction with the IVL promoter, but rather by recently described other mechanisms. For instance, the DEK protein was found to be transcriptionally up-regulated by HPV 16 E7, and this was shown to be important in the induction of cell proliferation and inhibition of the epithelial differentiation program [31, 32]. Furthermore, nucleophosmin (NPM) was reported to be up-regulated by HPV 16 E7 at the posttranscriptional level, and this up-regulation was suggested to have a role in the inhibition of differentiation in keratinocytes .
Both the DRR and the PRR of the human IVL gene contains binding sites for AP1 transcription factors (PRR). The promoters of differentiation-associated keratinocyte genes usually contain binding sites for the AP1 factors, and these are thought to be important in the regulation of gene expression by differentiation stimuli [25, 29]. It is also interesting to note that the HPV 16 E7 protein was shown to bind to AP1 transcription factors, including c-jun, junB, junD and c-fos . Therefore, we suppose that the AP1 motifs in the promoter of the human IVL gene may have a role in the regulation of gene expression by HPV oncoproteins. In order to prove this hypothesis, further research will be required using promoter mutagenesis and chromatin immunoprecipitation (ChIP) assays. It would be also interesting to study the effects of the HPV oncogenes on the expression of other genes (such as keratins, small prolin-rich proteins, S100 calcium binding proteins) involved in keratinocyte differentiation.
The decreased expression of IVL and other differentiation-regulated genes by the HPV oncoproteins may have an important role in the productive life cycle of the virus. HPV replication takes place in differentiating epithelial cells, which would exit the cell cycle in the absence of viral infection. The E7 oncogene is able to induce the progression of the cell cycle in differentiating keratinocytes, which is important for viral DNA replication [6, 7]. On the other hand, the ability of the E6 oncogene to cause a delay in the induction of epithelial differentiation may also have a role in providing a cellular environment that is favourable for HPV replication. Our results indicate that one possible mechanism of the inhibition of keratinocyte differentiation by E6 may be the down-regulation of promoters of certain differentiation-regulated genes.