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HPV16 E5 deregulates the autophagic process in human keratinocytes.

Belleudi F, Nanni M, Raffa S, Torrisi MR - Oncotarget (2015)

Bottom Line: Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein.The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy.In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy.

ABSTRACT
Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

No MeSH data available.


Related in: MedlinePlus

Impairment of autophagy in cells stably expressing 16E5(a) HaCaT pMSG and HaCaT pMSG E5 cells were left untreated (0 h) or treated with Dex for 12 h or 24 h. The 16E5 the increasing mRNA transcript levels were quantitated by real-time relative RT-PCR and normalized with respect to those detected in W12p6 cells. (b) Cells were kept in complete medium or either serum-starved or stimulated with KGF for 24 h in presence or absence of Dex induction. Western blot analysis shows that in serum-kept cells (left panel) the very weak band corresponding to LC3-II is decreased at 12 h and 24 h of Dex treatment in HaCaT pMSG E5 cells, while no changes in the band intensity are observed in HaCaT pMSG cells. Upon serum starvation (middle panel) or KGF stimulation (right panel) the evident increase of LC3-II band is abolished by Dex treatment only in HaCaT pMSG E5 cells, but not in control cells. In absence of Dex treatment, the increase of LC3-II protein induced KGF is lower in HaCaT pMSG E5 than in control cells. The densitometric analysis and Student t test were performed as above: *, **p < 0.05 vs the corresponding Dex-untreated cells, • p < 0.05 vs the corresponding serum-cultured cells, •• NS vs the corresponding Dex-untreated cells, ••• NS vs the corresponding HaCaT pMSG cells, ••••, ••••• p < 0.05 vs the corresponding Dex-untreated cells, ^, ^^p < 0.05 vs the corresponding KGF-unstimulated cells, ♦♦♦NS vs the corresponding Dex-untreated cells, ^^^p < 0.01 vs the corresponding KGF-unstimulated cells, ^^^^p < 0.05 vs the corresponding Dex-untreated cells, ^^^^^p < 0.01 vs the corresponding Dex-untreated cells, ♦♦♦p < 0.05 vs the corresponding HaCaT pMSG cells. (c) Ultrastructural analysis of HaCaT pMSG and HaCaT pMSG E5 cells stimulated with KGF for 24 h in presence of Dex: the number of double-membrane autophagic vacuoles (asterisks) is lower in HaCaT pMSG E5 (right panel) compared to HaCaT pMSG cells (left and middle panels). ER, endoplasmic reticulum; M, mitochondrion; NM, nuclear membrane; PM, plasma membrane; G, Golgi complex. Bars: 0.5 μm.
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Figure 5: Impairment of autophagy in cells stably expressing 16E5(a) HaCaT pMSG and HaCaT pMSG E5 cells were left untreated (0 h) or treated with Dex for 12 h or 24 h. The 16E5 the increasing mRNA transcript levels were quantitated by real-time relative RT-PCR and normalized with respect to those detected in W12p6 cells. (b) Cells were kept in complete medium or either serum-starved or stimulated with KGF for 24 h in presence or absence of Dex induction. Western blot analysis shows that in serum-kept cells (left panel) the very weak band corresponding to LC3-II is decreased at 12 h and 24 h of Dex treatment in HaCaT pMSG E5 cells, while no changes in the band intensity are observed in HaCaT pMSG cells. Upon serum starvation (middle panel) or KGF stimulation (right panel) the evident increase of LC3-II band is abolished by Dex treatment only in HaCaT pMSG E5 cells, but not in control cells. In absence of Dex treatment, the increase of LC3-II protein induced KGF is lower in HaCaT pMSG E5 than in control cells. The densitometric analysis and Student t test were performed as above: *, **p < 0.05 vs the corresponding Dex-untreated cells, • p < 0.05 vs the corresponding serum-cultured cells, •• NS vs the corresponding Dex-untreated cells, ••• NS vs the corresponding HaCaT pMSG cells, ••••, ••••• p < 0.05 vs the corresponding Dex-untreated cells, ^, ^^p < 0.05 vs the corresponding KGF-unstimulated cells, ♦♦♦NS vs the corresponding Dex-untreated cells, ^^^p < 0.01 vs the corresponding KGF-unstimulated cells, ^^^^p < 0.05 vs the corresponding Dex-untreated cells, ^^^^^p < 0.01 vs the corresponding Dex-untreated cells, ♦♦♦p < 0.05 vs the corresponding HaCaT pMSG cells. (c) Ultrastructural analysis of HaCaT pMSG and HaCaT pMSG E5 cells stimulated with KGF for 24 h in presence of Dex: the number of double-membrane autophagic vacuoles (asterisks) is lower in HaCaT pMSG E5 (right panel) compared to HaCaT pMSG cells (left and middle panels). ER, endoplasmic reticulum; M, mitochondrion; NM, nuclear membrane; PM, plasma membrane; G, Golgi complex. Bars: 0.5 μm.

Mentions: In order to define whether the effect of 16E5 on autophagy could be dose-dependent, we took advantage of the use of HaCaT cells stably transfected with the construct pMSG 16E5 (HaCaT pMSG E5) [22], in which the expression of the viral protein can be progressively induced, in a time-dependent manner, by treatment with dexamethasone (Dex). The HaCaT pMSG cells were used as negative control. Cells were left untreated (0 h) or treated with Dex for 12 h or 24 h, and the increasing 16E5 mRNA transcript levels were quantitated by real-time relative RT-PCR. The mRNA amounts were normalized respect to the levels expressed in W12p6 cells. The results clearly indicated that in HaCaT pMSG E5 cells, which expressed very low levels of 16E5 mRNA also in absence of Dex treatment [22–24], the increasing levels of 16E5 mRNA after Dex stimulation remain lower than those observed in the endogenous model of W12p6 cells (Figure 5a). To first analyse the impact of the progressive expression of 16E5 on basal autophagy, cells were kept in complete medium and treated with Dex as above. Western blot analysis showed that in HaCaT pMSG E5 cells the low expression of LC3-II protein was decreased already after 12 h of Dex treatment and no further decrease was observed after 24 h (Figure 5b, left panel). Interestingly, no changes on LC3-II amounts were induced by Dex in control cells (Figure 5b, left panel), demonstrating that the inhibitory effect observed in HaCaT pMSG E5 cells can be specifically ascribed to 16E5 expression. In addition, these results indicate that the observed inhibition of autophagy does not occur only when the viral protein is overexpressed.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

Belleudi F, Nanni M, Raffa S, Torrisi MR - Oncotarget (2015)

Impairment of autophagy in cells stably expressing 16E5(a) HaCaT pMSG and HaCaT pMSG E5 cells were left untreated (0 h) or treated with Dex for 12 h or 24 h. The 16E5 the increasing mRNA transcript levels were quantitated by real-time relative RT-PCR and normalized with respect to those detected in W12p6 cells. (b) Cells were kept in complete medium or either serum-starved or stimulated with KGF for 24 h in presence or absence of Dex induction. Western blot analysis shows that in serum-kept cells (left panel) the very weak band corresponding to LC3-II is decreased at 12 h and 24 h of Dex treatment in HaCaT pMSG E5 cells, while no changes in the band intensity are observed in HaCaT pMSG cells. Upon serum starvation (middle panel) or KGF stimulation (right panel) the evident increase of LC3-II band is abolished by Dex treatment only in HaCaT pMSG E5 cells, but not in control cells. In absence of Dex treatment, the increase of LC3-II protein induced KGF is lower in HaCaT pMSG E5 than in control cells. The densitometric analysis and Student t test were performed as above: *, **p < 0.05 vs the corresponding Dex-untreated cells, • p < 0.05 vs the corresponding serum-cultured cells, •• NS vs the corresponding Dex-untreated cells, ••• NS vs the corresponding HaCaT pMSG cells, ••••, ••••• p < 0.05 vs the corresponding Dex-untreated cells, ^, ^^p < 0.05 vs the corresponding KGF-unstimulated cells, ♦♦♦NS vs the corresponding Dex-untreated cells, ^^^p < 0.01 vs the corresponding KGF-unstimulated cells, ^^^^p < 0.05 vs the corresponding Dex-untreated cells, ^^^^^p < 0.01 vs the corresponding Dex-untreated cells, ♦♦♦p < 0.05 vs the corresponding HaCaT pMSG cells. (c) Ultrastructural analysis of HaCaT pMSG and HaCaT pMSG E5 cells stimulated with KGF for 24 h in presence of Dex: the number of double-membrane autophagic vacuoles (asterisks) is lower in HaCaT pMSG E5 (right panel) compared to HaCaT pMSG cells (left and middle panels). ER, endoplasmic reticulum; M, mitochondrion; NM, nuclear membrane; PM, plasma membrane; G, Golgi complex. Bars: 0.5 μm.
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Figure 5: Impairment of autophagy in cells stably expressing 16E5(a) HaCaT pMSG and HaCaT pMSG E5 cells were left untreated (0 h) or treated with Dex for 12 h or 24 h. The 16E5 the increasing mRNA transcript levels were quantitated by real-time relative RT-PCR and normalized with respect to those detected in W12p6 cells. (b) Cells were kept in complete medium or either serum-starved or stimulated with KGF for 24 h in presence or absence of Dex induction. Western blot analysis shows that in serum-kept cells (left panel) the very weak band corresponding to LC3-II is decreased at 12 h and 24 h of Dex treatment in HaCaT pMSG E5 cells, while no changes in the band intensity are observed in HaCaT pMSG cells. Upon serum starvation (middle panel) or KGF stimulation (right panel) the evident increase of LC3-II band is abolished by Dex treatment only in HaCaT pMSG E5 cells, but not in control cells. In absence of Dex treatment, the increase of LC3-II protein induced KGF is lower in HaCaT pMSG E5 than in control cells. The densitometric analysis and Student t test were performed as above: *, **p < 0.05 vs the corresponding Dex-untreated cells, • p < 0.05 vs the corresponding serum-cultured cells, •• NS vs the corresponding Dex-untreated cells, ••• NS vs the corresponding HaCaT pMSG cells, ••••, ••••• p < 0.05 vs the corresponding Dex-untreated cells, ^, ^^p < 0.05 vs the corresponding KGF-unstimulated cells, ♦♦♦NS vs the corresponding Dex-untreated cells, ^^^p < 0.01 vs the corresponding KGF-unstimulated cells, ^^^^p < 0.05 vs the corresponding Dex-untreated cells, ^^^^^p < 0.01 vs the corresponding Dex-untreated cells, ♦♦♦p < 0.05 vs the corresponding HaCaT pMSG cells. (c) Ultrastructural analysis of HaCaT pMSG and HaCaT pMSG E5 cells stimulated with KGF for 24 h in presence of Dex: the number of double-membrane autophagic vacuoles (asterisks) is lower in HaCaT pMSG E5 (right panel) compared to HaCaT pMSG cells (left and middle panels). ER, endoplasmic reticulum; M, mitochondrion; NM, nuclear membrane; PM, plasma membrane; G, Golgi complex. Bars: 0.5 μm.
Mentions: In order to define whether the effect of 16E5 on autophagy could be dose-dependent, we took advantage of the use of HaCaT cells stably transfected with the construct pMSG 16E5 (HaCaT pMSG E5) [22], in which the expression of the viral protein can be progressively induced, in a time-dependent manner, by treatment with dexamethasone (Dex). The HaCaT pMSG cells were used as negative control. Cells were left untreated (0 h) or treated with Dex for 12 h or 24 h, and the increasing 16E5 mRNA transcript levels were quantitated by real-time relative RT-PCR. The mRNA amounts were normalized respect to the levels expressed in W12p6 cells. The results clearly indicated that in HaCaT pMSG E5 cells, which expressed very low levels of 16E5 mRNA also in absence of Dex treatment [22–24], the increasing levels of 16E5 mRNA after Dex stimulation remain lower than those observed in the endogenous model of W12p6 cells (Figure 5a). To first analyse the impact of the progressive expression of 16E5 on basal autophagy, cells were kept in complete medium and treated with Dex as above. Western blot analysis showed that in HaCaT pMSG E5 cells the low expression of LC3-II protein was decreased already after 12 h of Dex treatment and no further decrease was observed after 24 h (Figure 5b, left panel). Interestingly, no changes on LC3-II amounts were induced by Dex in control cells (Figure 5b, left panel), demonstrating that the inhibitory effect observed in HaCaT pMSG E5 cells can be specifically ascribed to 16E5 expression. In addition, these results indicate that the observed inhibition of autophagy does not occur only when the viral protein is overexpressed.

Bottom Line: Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein.The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy.In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy.

ABSTRACT
Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

No MeSH data available.


Related in: MedlinePlus