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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 depletion in the W12p6 cervical carcinogenesis model restores the autophagic gene expression(a, b) HKs pCI-neo and HKs 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 genes (b). (c, d) W12p6 control siRNA and W12p6 E5 siRNA cells and HKs were treated as above. Real-time relative RT-PCR of key regulatory autophagy genes (c) or p53-target genes (d). Results are expressed as mean ± standard error (SE). Student t test was performed and significance levels have been defined as above: (a) *p < 0.05 and **** and ^^p < 0.005 vs the corresponding KGF-unstimulated cells, **p < 0.05, ^p < 0.005 and ***, *****, ^^^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (b) *, **, ***, ^^p < 0.05 and ^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (c) *, **p < 0.001 and ***, ****p < 0.05 vs the corresponding W12p6 control siRNA, NS vs the corresponding W12p6 control siRNA; (d) *, **, ***, ^^p < 0.05 and ****, *****, ^p < 0.005 vs the corresponding W12p6 control siRNA.
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Figure 8: 16E5 depletion in the W12p6 cervical carcinogenesis model restores the autophagic gene expression(a, b) HKs pCI-neo and HKs 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 genes (b). (c, d) W12p6 control siRNA and W12p6 E5 siRNA cells and HKs were treated as above. Real-time relative RT-PCR of key regulatory autophagy genes (c) or p53-target genes (d). Results are expressed as mean ± standard error (SE). Student t test was performed and significance levels have been defined as above: (a) *p < 0.05 and **** and ^^p < 0.005 vs the corresponding KGF-unstimulated cells, **p < 0.05, ^p < 0.005 and ***, *****, ^^^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (b) *, **, ***, ^^p < 0.05 and ^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (c) *, **p < 0.001 and ***, ****p < 0.05 vs the corresponding W12p6 control siRNA, NS vs the corresponding W12p6 control siRNA; (d) *, **, ***, ^^p < 0.05 and ****, *****, ^p < 0.005 vs the corresponding W12p6 control siRNA.

Mentions: In order to verify if the ability of 16E5 to transcriptionally regulate autophagy is a general phenomenon, we examined the expression of the autophagic genes in primary human keratinocytes transiently transfected with 16E5 (HKs E5) or with the pCI-neo empty vector (HKs pCI-neo) as control. The results showed that, also in primary cultures, the expression of 16E5 appeared to down-regulate most of the p53-independent (Figure 8a) and p53-regulated (Figure 8b) autophagy genes, as well as that of the main p53-target gene p21 (Figure 8b). Consistently with the results obtained in HaCaT cells, also in HKs E5 cells the stimulation with KGF significantly increased the expression of BECN1, ATG5 and LC3, while p53-target genes appeared unaffected (Figure 8a, 8b), confirming that KGF appears to exert a transcriptional control only on the p53-independent autophagy genes.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

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

16E5 depletion in the W12p6 cervical carcinogenesis model restores the autophagic gene expression(a, b) HKs pCI-neo and HKs 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 genes (b). (c, d) W12p6 control siRNA and W12p6 E5 siRNA cells and HKs were treated as above. Real-time relative RT-PCR of key regulatory autophagy genes (c) or p53-target genes (d). Results are expressed as mean ± standard error (SE). Student t test was performed and significance levels have been defined as above: (a) *p < 0.05 and **** and ^^p < 0.005 vs the corresponding KGF-unstimulated cells, **p < 0.05, ^p < 0.005 and ***, *****, ^^^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (b) *, **, ***, ^^p < 0.05 and ^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (c) *, **p < 0.001 and ***, ****p < 0.05 vs the corresponding W12p6 control siRNA, NS vs the corresponding W12p6 control siRNA; (d) *, **, ***, ^^p < 0.05 and ****, *****, ^p < 0.005 vs the corresponding W12p6 control siRNA.
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Figure 8: 16E5 depletion in the W12p6 cervical carcinogenesis model restores the autophagic gene expression(a, b) HKs pCI-neo and HKs 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 genes (b). (c, d) W12p6 control siRNA and W12p6 E5 siRNA cells and HKs were treated as above. Real-time relative RT-PCR of key regulatory autophagy genes (c) or p53-target genes (d). Results are expressed as mean ± standard error (SE). Student t test was performed and significance levels have been defined as above: (a) *p < 0.05 and **** and ^^p < 0.005 vs the corresponding KGF-unstimulated cells, **p < 0.05, ^p < 0.005 and ***, *****, ^^^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (b) *, **, ***, ^^p < 0.05 and ^p < 0.001 vs the corresponding HKs pCI-neo cells, NS vs the corresponding HKs pCI-neo cells; (c) *, **p < 0.001 and ***, ****p < 0.05 vs the corresponding W12p6 control siRNA, NS vs the corresponding W12p6 control siRNA; (d) *, **, ***, ^^p < 0.05 and ****, *****, ^p < 0.005 vs the corresponding W12p6 control siRNA.
Mentions: In order to verify if the ability of 16E5 to transcriptionally regulate autophagy is a general phenomenon, we examined the expression of the autophagic genes in primary human keratinocytes transiently transfected with 16E5 (HKs E5) or with the pCI-neo empty vector (HKs pCI-neo) as control. The results showed that, also in primary cultures, the expression of 16E5 appeared to down-regulate most of the p53-independent (Figure 8a) and p53-regulated (Figure 8b) autophagy genes, as well as that of the main p53-target gene p21 (Figure 8b). Consistently with the results obtained in HaCaT cells, also in HKs E5 cells the stimulation with KGF significantly increased the expression of BECN1, ATG5 and LC3, while p53-target genes appeared unaffected (Figure 8a, 8b), confirming that KGF appears to exert a transcriptional control only on the p53-independent autophagy genes.

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