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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

16E5 inhibits autophagosome assembly(a) HaCaT mCherry-EGFP-LC3 and HaCaT mCherry-EGFP-LC3/E5 cells were serum-starved or treated with KGF as above. Immunofluorescence analysis shows that in E5 expressing cells the number of yellow dots corresponding to newly assembled autophagosomes is decreased, while the red dots corresponding to autolysosomes are not increased compared to control cells. The quantitative analysis and Student t test were performed as above: *p < 0.05, **p < 0.01 vs the corresponding HaCaT mCherry-EGFP-LC3 cells. Bar: 10 μm (b) HaCaT pCIneo and HaCaT pCI-neo/E5 cells were serum-starved or treated with KGF in the presence or absence of leupeptin (LEU) as reported in materials and methods. Western blot shows that in 16E5 expressing cells the LC3-II levels are significantly reduced also in the presence of the inhibitor of the lysosomal degradation. The densitometric analysis and Student t test were performed as reported above: * and **p < 0.05 vs the corresponding HaCaT pCI-neo cells, *** and ****p < 0.01 vs the corresponding HaCaT pCI-neo cells.
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Figure 4: 16E5 inhibits autophagosome assembly(a) HaCaT mCherry-EGFP-LC3 and HaCaT mCherry-EGFP-LC3/E5 cells were serum-starved or treated with KGF as above. Immunofluorescence analysis shows that in E5 expressing cells the number of yellow dots corresponding to newly assembled autophagosomes is decreased, while the red dots corresponding to autolysosomes are not increased compared to control cells. The quantitative analysis and Student t test were performed as above: *p < 0.05, **p < 0.01 vs the corresponding HaCaT mCherry-EGFP-LC3 cells. Bar: 10 μm (b) HaCaT pCIneo and HaCaT pCI-neo/E5 cells were serum-starved or treated with KGF in the presence or absence of leupeptin (LEU) as reported in materials and methods. Western blot shows that in 16E5 expressing cells the LC3-II levels are significantly reduced also in the presence of the inhibitor of the lysosomal degradation. The densitometric analysis and Student t test were performed as reported above: * and **p < 0.05 vs the corresponding HaCaT pCI-neo cells, *** and ****p < 0.01 vs the corresponding HaCaT pCI-neo cells.

Mentions: In order to confirm that 16E5 is able to impact the autophagy on-rate, rather than the autophagy off-rate, as already indicated above by SQSTM1 monitoring, immunofluorescence experiments were performed doubly transfecting HaCaT cells with 16E5 and a pDest-mCherry-EGFP-LC3 tandem construct [20]. In fact, mCherry-EGFP-LC3 is an autophagic flux sensor, since EGFP fluorescence is quenched in acidic environments, whereas mCherry is an acidic-stable fluorescent tag: the nascent autophagosomes are both red and green (yellow) labeled, whereas the acidic autolysosomes appear red, as a consequence of the EGFP quenching. Quantitative immunofluorescence analysis performed upon either serum deprivation and KGF stimulation showed that 16E5 expression led to a significant decrease in the number of yellow dots per cells corresponding to newly assembled autophagosomes (Figure 4a), while the quantity of red dots corresponding to autophagosomes flowed in the lysosomes was not affected (Figure 4a). The inhibitory effect of 16E5 on autophagosome formation was further confirmed monitoring the LC3-II levels in presence or absence of the well known lysosomal protease inhibitor leupeptin (LEU, Figure 4b), which inhibits the vacuolar type H+-ATPase (v-ATPase) complex necessary for lysosomal acidification [21]. Western blot analysis performed upon serum deprivation or KGF stimulation showed that 16E5 expression significantly decreases LC3-II levels also in the presence of the inhibitor of the autophagic flux (Figure 4b), confirming that, independently from the stimulus which triggers autophagy, 16E5 exerts an inhibitory role in the autophagosome assembly.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

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

16E5 inhibits autophagosome assembly(a) HaCaT mCherry-EGFP-LC3 and HaCaT mCherry-EGFP-LC3/E5 cells were serum-starved or treated with KGF as above. Immunofluorescence analysis shows that in E5 expressing cells the number of yellow dots corresponding to newly assembled autophagosomes is decreased, while the red dots corresponding to autolysosomes are not increased compared to control cells. The quantitative analysis and Student t test were performed as above: *p < 0.05, **p < 0.01 vs the corresponding HaCaT mCherry-EGFP-LC3 cells. Bar: 10 μm (b) HaCaT pCIneo and HaCaT pCI-neo/E5 cells were serum-starved or treated with KGF in the presence or absence of leupeptin (LEU) as reported in materials and methods. Western blot shows that in 16E5 expressing cells the LC3-II levels are significantly reduced also in the presence of the inhibitor of the lysosomal degradation. The densitometric analysis and Student t test were performed as reported above: * and **p < 0.05 vs the corresponding HaCaT pCI-neo cells, *** and ****p < 0.01 vs the corresponding HaCaT pCI-neo cells.
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Figure 4: 16E5 inhibits autophagosome assembly(a) HaCaT mCherry-EGFP-LC3 and HaCaT mCherry-EGFP-LC3/E5 cells were serum-starved or treated with KGF as above. Immunofluorescence analysis shows that in E5 expressing cells the number of yellow dots corresponding to newly assembled autophagosomes is decreased, while the red dots corresponding to autolysosomes are not increased compared to control cells. The quantitative analysis and Student t test were performed as above: *p < 0.05, **p < 0.01 vs the corresponding HaCaT mCherry-EGFP-LC3 cells. Bar: 10 μm (b) HaCaT pCIneo and HaCaT pCI-neo/E5 cells were serum-starved or treated with KGF in the presence or absence of leupeptin (LEU) as reported in materials and methods. Western blot shows that in 16E5 expressing cells the LC3-II levels are significantly reduced also in the presence of the inhibitor of the lysosomal degradation. The densitometric analysis and Student t test were performed as reported above: * and **p < 0.05 vs the corresponding HaCaT pCI-neo cells, *** and ****p < 0.01 vs the corresponding HaCaT pCI-neo cells.
Mentions: In order to confirm that 16E5 is able to impact the autophagy on-rate, rather than the autophagy off-rate, as already indicated above by SQSTM1 monitoring, immunofluorescence experiments were performed doubly transfecting HaCaT cells with 16E5 and a pDest-mCherry-EGFP-LC3 tandem construct [20]. In fact, mCherry-EGFP-LC3 is an autophagic flux sensor, since EGFP fluorescence is quenched in acidic environments, whereas mCherry is an acidic-stable fluorescent tag: the nascent autophagosomes are both red and green (yellow) labeled, whereas the acidic autolysosomes appear red, as a consequence of the EGFP quenching. Quantitative immunofluorescence analysis performed upon either serum deprivation and KGF stimulation showed that 16E5 expression led to a significant decrease in the number of yellow dots per cells corresponding to newly assembled autophagosomes (Figure 4a), while the quantity of red dots corresponding to autophagosomes flowed in the lysosomes was not affected (Figure 4a). The inhibitory effect of 16E5 on autophagosome formation was further confirmed monitoring the LC3-II levels in presence or absence of the well known lysosomal protease inhibitor leupeptin (LEU, Figure 4b), which inhibits the vacuolar type H+-ATPase (v-ATPase) complex necessary for lysosomal acidification [21]. Western blot analysis performed upon serum deprivation or KGF stimulation showed that 16E5 expression significantly decreases LC3-II levels also in the presence of the inhibitor of the autophagic flux (Figure 4b), confirming that, independently from the stimulus which triggers autophagy, 16E5 exerts an inhibitory role in the autophagosome assembly.

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