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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

The inhibitory effect of 16E5 on KGF-triggered autophagy depends on KGFR expression and signaling(a) HaCaT cells were transfected with 16E5 cDNA (HaCaT E5), with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or with an unrelated siRNA (HaCaT control siRNA) as control. Alternatively cells were cotransfected with KGFRwt cDNA and with KGFR si RNA. Cells were then stimulated with KGF as above. Western blot analysis shows that, upon KGF stimulation, both KGFR and LC3-II bands are reduced, while the SQSTM1 band is increased either in 16E5-transfected and KGFR-depleted cells. (b) Cells were transiently transfected with 16E5 (HaCaT E5) or cotransfected with 16E5 and pCI-neo vector containing human KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−) and stimulated with KGF as above. Western blot analysis shows that the decrease of LC3-II as well as the increase of SQSTM1 induced by 16E5 expression is counteracted only by KGFRwt overexpression. The densitometric analysis and Student t test were performed as reported above: (a) ^, ^^^, ***p < 0.05 and *p < 0.01 vs the corrisponding HaCaT control siRNA cells, ^^p < 0.05 and **p < 0.01 vs the corrisponding HaCaT KGFR siRNA cells; (b) *, ^p < 0.05 vs HaCaT E5 cells.
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Figure 2: The inhibitory effect of 16E5 on KGF-triggered autophagy depends on KGFR expression and signaling(a) HaCaT cells were transfected with 16E5 cDNA (HaCaT E5), with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or with an unrelated siRNA (HaCaT control siRNA) as control. Alternatively cells were cotransfected with KGFRwt cDNA and with KGFR si RNA. Cells were then stimulated with KGF as above. Western blot analysis shows that, upon KGF stimulation, both KGFR and LC3-II bands are reduced, while the SQSTM1 band is increased either in 16E5-transfected and KGFR-depleted cells. (b) Cells were transiently transfected with 16E5 (HaCaT E5) or cotransfected with 16E5 and pCI-neo vector containing human KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−) and stimulated with KGF as above. Western blot analysis shows that the decrease of LC3-II as well as the increase of SQSTM1 induced by 16E5 expression is counteracted only by KGFRwt overexpression. The densitometric analysis and Student t test were performed as reported above: (a) ^, ^^^, ***p < 0.05 and *p < 0.01 vs the corrisponding HaCaT control siRNA cells, ^^p < 0.05 and **p < 0.01 vs the corrisponding HaCaT KGFR siRNA cells; (b) *, ^p < 0.05 vs HaCaT E5 cells.

Mentions: To clarify whether the inhibition of KGF-dependent autophagy induced by 16E5 is directly related to its previously reported ability to down-regulate KGFR expression and signaling [12, 13], we first compared the effects of 16E5 expression to those induced by KGFR depletion. HaCaT cells were singly transfected with 16E5 cDNA or with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or an unrelated siRNA (HaCaT control siRNA) as control and then stimulated with KGF as above. In addition, in order to assess whether the possible effects induced by KGFR depletion can be counteracted by its simultaneous forced expression, cells were also doubly transfected with KGFR siRNA and pCI-neo vector containing human KGFRwt (HaCaT KGFRwt cDNA/KGFR siRNA). Western blot analysis showed that both 16E5-transfected and KGFR-depleted cells not only displayed receptor down-regulation as expected [13], but also a significant decrease of LC3-II levels as well as a block of SQSTM1 degradation in response to KGF (Figure 2a). Moreover, the inhibitory effects on autophagy induced by KGFR depletion was reverted by the simultaneous overexpression of the receptor (Figure 2a). Thus, 16E5 expression and KGFR silencing appeared to affect the autophagic process in a similar manner. To further demonstrate the receptor involvement on the 16E5 effect on autophagy, we performed KGFR forced overexpression in the presence of the viral protein: to this aim, cells were transiently cotransfected with 16E5 (HaCaT E5) and KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−). After transfection, cells were stimulated with KGF as above. Western blot analysis clearly showed that the 16E5-induced decrease of LC3-II levels as well as SQSTM1 accumulation was reverted by the expression of KGFRwt, but not by that of KGFRkin- (Figure 2b). Therefore, KGFR forced expression and receptor activation are sufficient to counteract the inhibitory effect of 16E5 on the autophagy upon growth factor treatment. These results demonstrate that, although the molecular mechanisms remain to be clarified, 16E5 appears to impact the pro-autophagic KGFR pathway through the down-regulation of the receptor.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

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

The inhibitory effect of 16E5 on KGF-triggered autophagy depends on KGFR expression and signaling(a) HaCaT cells were transfected with 16E5 cDNA (HaCaT E5), with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or with an unrelated siRNA (HaCaT control siRNA) as control. Alternatively cells were cotransfected with KGFRwt cDNA and with KGFR si RNA. Cells were then stimulated with KGF as above. Western blot analysis shows that, upon KGF stimulation, both KGFR and LC3-II bands are reduced, while the SQSTM1 band is increased either in 16E5-transfected and KGFR-depleted cells. (b) Cells were transiently transfected with 16E5 (HaCaT E5) or cotransfected with 16E5 and pCI-neo vector containing human KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−) and stimulated with KGF as above. Western blot analysis shows that the decrease of LC3-II as well as the increase of SQSTM1 induced by 16E5 expression is counteracted only by KGFRwt overexpression. The densitometric analysis and Student t test were performed as reported above: (a) ^, ^^^, ***p < 0.05 and *p < 0.01 vs the corrisponding HaCaT control siRNA cells, ^^p < 0.05 and **p < 0.01 vs the corrisponding HaCaT KGFR siRNA cells; (b) *, ^p < 0.05 vs HaCaT E5 cells.
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Figure 2: The inhibitory effect of 16E5 on KGF-triggered autophagy depends on KGFR expression and signaling(a) HaCaT cells were transfected with 16E5 cDNA (HaCaT E5), with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or with an unrelated siRNA (HaCaT control siRNA) as control. Alternatively cells were cotransfected with KGFRwt cDNA and with KGFR si RNA. Cells were then stimulated with KGF as above. Western blot analysis shows that, upon KGF stimulation, both KGFR and LC3-II bands are reduced, while the SQSTM1 band is increased either in 16E5-transfected and KGFR-depleted cells. (b) Cells were transiently transfected with 16E5 (HaCaT E5) or cotransfected with 16E5 and pCI-neo vector containing human KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−) and stimulated with KGF as above. Western blot analysis shows that the decrease of LC3-II as well as the increase of SQSTM1 induced by 16E5 expression is counteracted only by KGFRwt overexpression. The densitometric analysis and Student t test were performed as reported above: (a) ^, ^^^, ***p < 0.05 and *p < 0.01 vs the corrisponding HaCaT control siRNA cells, ^^p < 0.05 and **p < 0.01 vs the corrisponding HaCaT KGFR siRNA cells; (b) *, ^p < 0.05 vs HaCaT E5 cells.
Mentions: To clarify whether the inhibition of KGF-dependent autophagy induced by 16E5 is directly related to its previously reported ability to down-regulate KGFR expression and signaling [12, 13], we first compared the effects of 16E5 expression to those induced by KGFR depletion. HaCaT cells were singly transfected with 16E5 cDNA or with a small interfering RNA for FGFR2/Bek (HaCaT KGFR siRNA) or an unrelated siRNA (HaCaT control siRNA) as control and then stimulated with KGF as above. In addition, in order to assess whether the possible effects induced by KGFR depletion can be counteracted by its simultaneous forced expression, cells were also doubly transfected with KGFR siRNA and pCI-neo vector containing human KGFRwt (HaCaT KGFRwt cDNA/KGFR siRNA). Western blot analysis showed that both 16E5-transfected and KGFR-depleted cells not only displayed receptor down-regulation as expected [13], but also a significant decrease of LC3-II levels as well as a block of SQSTM1 degradation in response to KGF (Figure 2a). Moreover, the inhibitory effects on autophagy induced by KGFR depletion was reverted by the simultaneous overexpression of the receptor (Figure 2a). Thus, 16E5 expression and KGFR silencing appeared to affect the autophagic process in a similar manner. To further demonstrate the receptor involvement on the 16E5 effect on autophagy, we performed KGFR forced overexpression in the presence of the viral protein: to this aim, cells were transiently cotransfected with 16E5 (HaCaT E5) and KGFRwt (HaCaT E5/KGFRwt) or the kinase negative mutant KGFRY656F/Y657F (HaCaT E5/KGFRkin−). After transfection, cells were stimulated with KGF as above. Western blot analysis clearly showed that the 16E5-induced decrease of LC3-II levels as well as SQSTM1 accumulation was reverted by the expression of KGFRwt, but not by that of KGFRkin- (Figure 2b). Therefore, KGFR forced expression and receptor activation are sufficient to counteract the inhibitory effect of 16E5 on the autophagy upon growth factor treatment. These results demonstrate that, although the molecular mechanisms remain to be clarified, 16E5 appears to impact the pro-autophagic KGFR pathway through the down-regulation of the receptor.

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