Limits...
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 expression down-modulates the autophagy gene expression in HaCaT cells(a, b) HaCaT pCI-neo and HaCaT E5 cells were kept in complete medium or serum-starved or stimulated with KGF as above. Real-time relative RT-PCR of key regulatory autophagy genes (a) or p53-target autophagic (ULK1, ULK2, ATG4a, ATG7) or autophagy-independent (p21, 14–3-3-σ) genes (b). Results are expressed as mean ± standard error (SE) from three different experiments in triplicate. Student t test was performed and significance levels have been defined as p < 0.05: (a) *p < 0.05 and **p < 0.01 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding HaCaT pCI-neo cells, ^p < 0.05 vs the corresponding KGF-unstimulated cells. (b) *, **, ^, ^^p < 0.05 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding KGF-unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496223&req=5

Figure 7: 16E5 expression down-modulates the autophagy gene expression in HaCaT cells(a, b) HaCaT pCI-neo and HaCaT E5 cells were kept in complete medium or serum-starved or stimulated with KGF as above. Real-time relative RT-PCR of key regulatory autophagy genes (a) or p53-target autophagic (ULK1, ULK2, ATG4a, ATG7) or autophagy-independent (p21, 14–3-3-σ) genes (b). Results are expressed as mean ± standard error (SE) from three different experiments in triplicate. Student t test was performed and significance levels have been defined as p < 0.05: (a) *p < 0.05 and **p < 0.01 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding HaCaT pCI-neo cells, ^p < 0.05 vs the corresponding KGF-unstimulated cells. (b) *, **, ^, ^^p < 0.05 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding KGF-unstimulated cells.

Mentions: Since it has been demonstrated that 16E5 is able to affect the expression of several host genes [25, 26] and growing evidences indicate that autophagy is not only post-translationally regulated, but also transcriptionally controlled [27–29], here we investigated whether 16E5 might interfere with autophagy by affecting the autophagic gene expression. To this aim, the mRNA transcript levels of different crucial autophagic genes acting at different steps of the process (BECN1, ATG5 and LC3) were estimated by real-time relative RT-PCR in HaCaT E5 cells and normalized respect to the levels detected in HaCaT pCI-neo cells. In cells kept in complete medium, BECN1 and ATG5, but not LC3 or ATG7, appeared down-regulated by 16E5 expression (Figure 7a, upper panels). Moreover, when autophagy is stimulated by serum starvation or KGF treatment, a drastic significant decreased expression of all genes examined, except BECN1 in serum-deprived cells, was evident (Figure 7a, lower panels). Thus, 16E5 down-regulates autophagy gene expression when the process is induced as well as under basal conditions. Interestingly, in agreement with our previous biochemical observations [16], KGF stimulation slightly but significantly increased the expression of BECN1 and LC3, while that of ATG5 seemed unaffected (Figure 7a, lower panels) indicating that KGF/KGFR signaling plays a role in the transcriptional control of autophagy.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

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

16E5 expression down-modulates the autophagy gene expression in HaCaT cells(a, b) HaCaT pCI-neo and HaCaT E5 cells were kept in complete medium or serum-starved or stimulated with KGF as above. Real-time relative RT-PCR of key regulatory autophagy genes (a) or p53-target autophagic (ULK1, ULK2, ATG4a, ATG7) or autophagy-independent (p21, 14–3-3-σ) genes (b). Results are expressed as mean ± standard error (SE) from three different experiments in triplicate. Student t test was performed and significance levels have been defined as p < 0.05: (a) *p < 0.05 and **p < 0.01 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding HaCaT pCI-neo cells, ^p < 0.05 vs the corresponding KGF-unstimulated cells. (b) *, **, ^, ^^p < 0.05 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding KGF-unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496223&req=5

Figure 7: 16E5 expression down-modulates the autophagy gene expression in HaCaT cells(a, b) HaCaT pCI-neo and HaCaT E5 cells were kept in complete medium or serum-starved or stimulated with KGF as above. Real-time relative RT-PCR of key regulatory autophagy genes (a) or p53-target autophagic (ULK1, ULK2, ATG4a, ATG7) or autophagy-independent (p21, 14–3-3-σ) genes (b). Results are expressed as mean ± standard error (SE) from three different experiments in triplicate. Student t test was performed and significance levels have been defined as p < 0.05: (a) *p < 0.05 and **p < 0.01 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding HaCaT pCI-neo cells, ^p < 0.05 vs the corresponding KGF-unstimulated cells. (b) *, **, ^, ^^p < 0.05 vs the corresponding HaCaT pCI-neo cells, NS vs the corresponding KGF-unstimulated cells.
Mentions: Since it has been demonstrated that 16E5 is able to affect the expression of several host genes [25, 26] and growing evidences indicate that autophagy is not only post-translationally regulated, but also transcriptionally controlled [27–29], here we investigated whether 16E5 might interfere with autophagy by affecting the autophagic gene expression. To this aim, the mRNA transcript levels of different crucial autophagic genes acting at different steps of the process (BECN1, ATG5 and LC3) were estimated by real-time relative RT-PCR in HaCaT E5 cells and normalized respect to the levels detected in HaCaT pCI-neo cells. In cells kept in complete medium, BECN1 and ATG5, but not LC3 or ATG7, appeared down-regulated by 16E5 expression (Figure 7a, upper panels). Moreover, when autophagy is stimulated by serum starvation or KGF treatment, a drastic significant decreased expression of all genes examined, except BECN1 in serum-deprived cells, was evident (Figure 7a, lower panels). Thus, 16E5 down-regulates autophagy gene expression when the process is induced as well as under basal conditions. Interestingly, in agreement with our previous biochemical observations [16], KGF stimulation slightly but significantly increased the expression of BECN1 and LC3, while that of ATG5 seemed unaffected (Figure 7a, lower panels) indicating that KGF/KGFR signaling plays a role in the transcriptional control of autophagy.

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