José Bonilla-Delgado

The E6 Oncoprotein from HPV16 Enhances the Canonical Wnt/β-catenin Pathway in Skin Epidermis in vivo.

The contribution of the Wnt signaling pathway to human papilloma virus (HPV)-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk HPVs (HR-HPV), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the PDZ-binding domain of E6 protein. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2+/LacZ mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellular β-catenin–responsive genes, and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2+/LacZ. Conversely, cotransfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that used a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only. Mol Cancer Res; 10(2); 250–8. ©2011 AACR.

EGF Regulates Claudin‐2 and ‐4 Expression Through Src and STAT3 in MDCK Cells

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2‐dependent downregulation of claudin‐2 (CLDN‐2) and upregulation of claudin‐4 (CLDN‐4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN‐2 downregulation and CLDN‐4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN‐2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF‐induced changes on CLDN‐2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF‐induced downregulation of CLDN‐2, but they are also implicated in the EGF‐induced CLDN‐4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN‐2 and ‐4 proteins and mRNAs through Src and STAT3 activity. J. Cell. Physiol. 230: 105–115, 2015.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue.

The HPV16 E7 oncoprotein and 17β-estradiol are important factors for the induction of premalignant lesions and cervical cancer. The study of these factors is crucial for a better understanding of cervical tumorigenesis. Here, we assessed the global gene expression profiles induced by the HPV16 E7 oncoprotein and/or 17β-estradiol in cervical tissue of FvB and K14E7 transgenic mice. We found that the most dramatic changes in gene expression occurred in K14E7 and FvB groups treated with 17β-estradiol. A large number of differentially expressed genes involved in the immune response were observed in 17β-estradiol treated groups. The E7 oncoprotein mainly affected the expression of genes involved in cellular metabolism. Our microarray data also identified differentially expressed genes that have not previously been reported in cervical cancer. The identification of genes regulated by E7 and 17β-estradiol, provides the basis for further studies on their role in cervical carcinogenesis.

Human Papillomavirus E6/E7 Oncogenes Promote Mouse Ear Regeneration by Increasing the Rate of Wound Re-epithelization and Epidermal Growth

Mammals have limited regeneration capacity. We report here that, in transgenic mice (Tg(bK6-E6/E7)), the expression of the E6/E7 oncogenes of human papilloma virus type 16 (HPV16) under the control of the bovine keratin 6 promoter markedly improves the mouses capacity to repair portions of the ear after being wounded. Increased repair capacity correlates with an increased number of epidermal proliferating cells. In concordance with the expected effects of the E6 and E7 oncogenes, levels of p53 decreased and those of p16 in epidermal cells increased. In addition, we observed that wound re-epithelization proceeded faster in transgenic than in wild-type animals. After the initial re-epithelization, epidermal cell migration from the intact surrounding tissue appears to be a major contributor to the growing epidermis, especially in the repairing tissue of transgenic mice. We also found that there is a significantly higher number of putative epidermal stem cells in Tg(bK6-E6/E7) than in wild-type mice. Remarkably, hair follicles and cartilage regenerated within the repaired ear tissue, without evidence of tumor formation. We propose that the ability to regenerate ear portions is limited by the capacity of the epidermis to repair itself and grow.

The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal

Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E2) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E2 on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E2 in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells.

The HPV16 E7 Oncoprotein Disrupts Dendritic Cell Function and Induces the Systemic Expansion of CD11b+Gr1+ Cells in a Transgenic Mouse Model

Objective. The aim of this study was to analyze the effects of the HPV16 E7 oncoprotein on dendritic cells (DCs) and CD11b+Gr1+ cells using the K14E7 transgenic mouse model. Materials and Methods. The morphology of DCs was analyzed in male mouse skin on epidermal sheets using immunofluorescence and confocal microscopy. Flow cytometry was used to determine the percentages of DCs and CD11b+Gr1+ cells in different tissues and to evaluate the migration of DCs. Results. In the K14E7 mouse model, the morphology of Langerhans cells and the migratory activity of dendritic cells were abnormal. An increase in CD11b+Gr1+ cells was observed in the blood and skin of K14E7 mice, and molecules related to CD11b+Gr1+ chemoattraction (MCP1 and S100A9) were upregulated. Conclusions. These data suggest that the HPV16 E7 oncoprotein impairs the function and morphology of DCs and induces the systemic accumulation of CD11b+Gr1+ cells.

Early synergistic interactions between the HPV16‑E7 oncoprotein and 17β-oestradiol for repressing the expression of Granzyme B in a cervical cancer model

Although high-risk human papillomavirus (HR-HPV) infection has a prominent role in the aetiology of cervical cancer (CC), sex steroid hormones may also be involved in this process; however, the cooperation between oestrogen and HR-HPV in the early stages of cervical carcinogenesis is poorly understood. Since 17β-oestradiol (E2) and the HPV type 16-E7 oncoprotein induce CC in transgenic mice, a microarray analysis was performed in the present study to generate global gene expression profiles from 2-month-old FVB (non-transgenic) and K14E7 (transgenic) mice who were left untreated or were treated for 1 month with E2. Upregulation of cancer-related genes that have not been previously reported in the context of CC, including glycerophosphodiester phosphodiesterase domain containing 3, interleukin 1 receptor type II, natriuretic peptide type C, MGAT4 family member C, lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) and glucoside xylosyltransferase 2, was observed. Notably, upregulation of the serine (or cysteine) peptidase inhibitor clade B member 9 gene and downregulation of the Granzyme gene family were observed; the repression of the Granzyme B pathway may be a novel mechanism of immune evasion by cancer cells. The present results provide the basis for further studies on early biomarkers of CC risk and synergistic interactions between HR-HPV and oestrogen.

The PDZ-Binding Motif of HPV16-E6 Oncoprotein Modulates the Keratinization and Stemness Transcriptional Profile In Vivo

Objective The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif. Materials and Methods The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence. Results The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151. Conclusion The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.